Polypeptides having protease activity for use in detergent compositions

Abstract

In the present invention, a novel protease is provided. More specifically, genetically engineered proteases, compositions comprising these enzymes, and methods of making and using these enzymes or compositions comprising these enzymes are provided.

Description

Technical Field

[0001] In the present invention, novel proteases are provided. More specifically, genetically engineered proteases, compositions comprising these enzymes, and methods for preparing and using these enzymes or compositions comprising these enzymes are provided. Background Art

[0002] Enzymes are increasingly used in various applications as sustainable alternatives to petrochemicals. Enzymes are biodegradable and already exhibit catalytic activity at lower temperatures, thereby reducing energy consumption. Specifically, in the detergent industry, enzymes are applied in washing formulations to improve cleaning efficiency and / or reduce energy consumption in the washing step.

[0003] Proteases are enzymes capable of hydrolyzing proteins. Thus, proteases have been used to remove protein stains and have been added to detergent compositions for this purpose. In detergent applications, proteases should be stable at elevated temperatures and / or within the denaturing conditions of detergents and washing solutions.

[0004] WO 2016 / 096711 and WO 2016 / 096714 describe subtilisin variants and detergents containing the variant, which have improved stability and / or improved washing performance in liquid detergents compared to the parental subtilisin. WO 2016 / 001450 and WO 2020 / 002255 disclose subtilisin variants with increased stability. WO 2010 / 056640 also describes subtilisin protease variants. US 6,376,450 discloses multiply substituted protease variants that provide improved and enhanced cleaning ability. US2020 / 172890 A1 discloses protease variants with enhanced performance and storage stability. DE 10 2018 004207A1 and WO 2018 / 069158 A1 also disclose protease variants.

[0005] Therefore, there is a need for new proteases that meet these requirements. Summary of the Invention

[0006] The present invention relates to variant polypeptides having protease activity or fragments of said polypeptides having protease activity, wherein:

[0007] (i) the polypeptide or its fragment has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and

[0008] (ii) Compared with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 and with reference to the numbering of SEQ ID NO:2, the polypeptide or its fragment contains amino acid substitutions at amino acid residues 43, 78 and 204.

[0009] The present invention further relates to polynucleotides encoding said variant polypeptides and compositions comprising said variant polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shows the residual activity (defined as the activity after storage divided by the activity at time point zero) of the variants of Table 5 (according to the presence of mutations N43K, S78N / D and N204D) after storage at 37 °C for 160 hours. DETAILED DESCRIPTION

[0011] The present invention can be more easily understood by reference to the following detailed description of the embodiments of the invention and the examples included herein.

[0012] Although the present invention will be described with respect to specific embodiments, the description should not be construed in a limiting sense.

[0013] Definition

[0014] Unless otherwise specified, the terms used herein should be understood according to the conventional usage of those of ordinary skill in the relevant art.

[0015] Before describing the exemplary embodiments of the present invention in detail, definitions important for understanding the present invention are given. Unless otherwise specified or obvious from the nature of the definition, the definitions apply to all compounds, methods and uses described herein.

[0016] As used in this specification and the appended claims, the singular forms "a / an" also include the corresponding plurals unless the context clearly dictates otherwise.

[0017] In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art will understand still ensures the technical effect of the feature being discussed. The term typically indicates a deviation from the indicated numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0018] In addition, the terms "first", "second", "third", or "(a)", "(b)", "(c)", "(d)", etc. in this specification and the claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments of the invention described herein are capable of operating in an order other than that described or illustrated herein. In the case where the terms "first", "second", "third", or "(a)", "(b)", "(c)", "(d)", "i", "ii", etc. relate to steps of a method or use or measurement, unless otherwise indicated in this application as described above or below, there is no temporal or time interval coherence between these steps, that is, these steps can be carried out simultaneously or there can be a time interval of seconds, minutes, hours, days, weeks, months, or even years between such steps.

[0019] Throughout this application, various publications are referenced. The disclosures of all these publications and those references cited in these publications are hereby incorporated by reference in their entirety into this application to more fully describe the prior art in the field to which the invention pertains.

[0020] It should be understood that the term "comprising" is not restrictive. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined hereinafter as including at least a certain number of members, this means that a group consisting only of these members is also covered.

[0021] A "parent" sequence (also referred to as "parent enzyme" or "parent protein") is the starting sequence used to introduce changes in a sequence (e.g., by introducing one or more amino acid substitutions) to generate a "variant" of the parent sequence. In the context of the present invention, the sequences according to any one of SEQ ID No: 1 and 3 to 11 and sequences having at least 60% identity thereto are considered parent sequences. The terms "enzyme variant" or "sequence variant" or "protein variant" or "variant polypeptide" or "protease variant" or "variant polypeptide having protease activity" are used interchangeably herein and are used with reference to the parent enzyme from which the corresponding variant enzyme is derived. Thus, parent enzymes include wild-type enzymes and variants of wild-type enzymes for the development of additional variants. Variant enzymes differ to some extent from the amino acid sequence of the parent enzyme.

[0022] When describing the variant polypeptides of the present invention, the abbreviations of individual amino acids are used according to the recognized IUPAC single-letter or three-letter amino acid abbreviations.

[0023] "Amino acid substitution" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the substituting amino acid. For example, substitution of histidine at position 120 by alanine is denoted as "His120Ala" or "H120A". Substitution can also be described without specifying the amino acid at that position in the parent, by naming only the resulting amino acid in the variant, e.g., by using "X120A" or "120A" or "Xaa120Ala" or "120Ala".

[0024] Variants containing multiple substitutions are separated by "+", e.g., "Arg170Tyr+Gly195Glu", "R170Y+G195E" or "X170Y+X195E" represent substitution of arginine and glycine at positions 170 and 195 by tyrosine and glutamate, respectively. Alternatively, multiple substitutions can be separated by a space or a comma, e.g., "R170Y G195E" or "R170Y,G195E". When different alternative substitutions can be introduced at a position, the different substitutions are separated by a comma, e.g., "Arg170Tyr,Glu" and "R170T,E" represent substitution of arginine at position 170 by tyrosine or glutamate, respectively. Alternative substitutions at a specific position can also be denoted as "X120A,G,H", "120A,G,H", "X120A / G / H" or "120A / G / H". Alternatively, different substitutions can be represented in parentheses, e.g., "Arg170[Tyr,Gly]" or "Arg170{Tyr,Gly}" or in short form as "R170[Y,G]" or "R170{Y,G}".

[0025] The numbering of the amino acid residues of the protease described herein is as is commonly used in the art for proteases (see P.N. Bryan, Biochimica et Biophysica Acta [Biochemistry and Biophysics Acta] 1543 (2000), 203 - 222, see column 1, paragraph 3 on page 204), according to the numbering of subtilisin BPN' 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 the "BPN' numbering").

[0026] Alternatively, amino acid positions can be described 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 ID 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:

[0027] Table 1

[0028]

[0029] The term "introducing at least two negative charges" into a specific amino acid sequence means that the net charge of the specific amino acid sequence is increased by at least two negative charges. Such an increase in the net charge of the specific amino acid sequence by at least two negative charges is achieved by altering the amino acid sequence and can be accomplished by one or more amino acid sequence alterations selected from the group consisting of substitution, deletion, and insertion, preferably by one or more amino acid substitutions. An increase in the net charge of the specific amino acid sequence by at least two negative charges can be achieved by removing positive charges and / or by introducing negative charges or a combination thereof. The four amino acids aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K), and arginine (Arg, R) have side chains that can be charged at neutral pH. At pH 7.0, two are negatively charged: aspartic acid (Asp, D) and glutamic acid (Glu, E) (acidic side chains), and two are positively charged: lysine (Lys, K) and arginine (Arg, R) (basic side chains). Thus, introducing at least two negative charges into an amino acid sequence can be accomplished, for example, by substituting arginine with glutamic acid, substituting two uncharged leucine residues with two glutamic acid residues, by inserting two aspartic acid residues, or by deleting two lysine residues. Evaluation of introducing at least two negative charges by amino acid sequence modification is preferably carried out under the conditions typically present in the washing step, preferably at pH 6 - 11, preferably at pH 7 - 9, more preferably at pH 7.5 - 8.5, further preferably at pH 7.0 - 8.0, and most preferably at pH 7.0 or pH 8.0. In a preferred embodiment of the present invention, at least two negative charges are introduced by substituting the arginine residue at position 101 (numbering according to SEQ ID NO:2) with aspartic acid or glutamic acid.

[0030] The term "native" (or natively or wild - type or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or polypeptide found in nature (i.e., without any human intervention).

[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 that is native to the host cell but has been structurally modified (e.g., deleted, substituted, and / or inserted) by recombinant DNA techniques to alter the native polypeptide; or a polypeptide that is native to the host cell but whose expression has been quantitatively altered or whose expression has been directed from a genomic location different from the native host cell due to manipulation of the host cell DNA by recombinant DNA techniques (e.g., a stronger promoter). Similarly, the term "heterologous" (or exogenous or foreign or recombinant or non-native or non-natural) polynucleotide refers to a polynucleotide that is not native to the host cell; a polynucleotide that is native to the host cell but has been structurally modified (e.g., deleted, substituted, and / or inserted) by recombinant DNA techniques to alter the native polynucleotide; or a polynucleotide that is native to the host cell but whose expression has been quantitatively altered due to manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques (e.g., a stronger promoter); or a polynucleotide that is native to the host cell but has not been integrated into its native genetic environment due to genetic manipulation by recombinant DNA techniques. For the relationship between two or more polynucleotide sequences or between two or more amino acid sequences, the term "heterologous" is used to characterize that two or more polynucleotide sequences or two or more amino acid sequences do not occur naturally in a particular combination with each other.

[0032] For the purposes of the present invention, "recombinant" (or transgenic) with respect to a cell or organism means that the cell or organism contains a heterologous polynucleotide introduced by a human using genetic technology. For a polynucleotide, "recombinant" includes all constructs produced by using genetic technology / recombinant DNA technology, wherein

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

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

[0035] (c) both a) and b)

[0036] is not in its wild-type genetic environment or has been artificially modified.

[0037] A "synthetic" compound is obtained by in vitro chemical and / or enzymatic synthesis.

[0038] Variant polynucleotide and variant polypeptide sequences can be defined by their sequence identity when compared to the parental sequence. Sequence identity is typically provided as "percent sequence identity" or "identity %". To calculate sequence identity, in a first step, a sequence alignment is generated. According to the present invention, a pairwise global alignment is generated, which means that the two sequences are aligned over their entire length, which is typically generated by using a mathematical method called an alignment algorithm.

[0039] According to the present invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. [Journal of Molecular Biology] (1979) 48, pp. 443-453). Preferably, the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the present invention, with the program default parameters (polynucleotide: gap open = 10.0, gap extend = 0.5, and matrix = EDNAFULL; polypeptide: gap open = 10.0, gap extend = 0.5, and matrix = EBLOSUM62). After aligning the two sequences, in a second step, the identity value is determined based on the generated alignment. For this purpose, the percent identity is calculated as follows: dividing the number of identical residues by the length of the alignment region of the corresponding sequence of the present invention over its entire length and multiplying by 100: percent identity = (identical residues / length of the alignment region of the corresponding sequence of the present invention over its entire length) * 100.

[0040] For calculating the percent identity of two nucleic acid sequences, the calculation and some explanations for percent identity of two amino acid sequences are equally applicable. For a nucleic acid sequence encoding a protein, the pairwise alignment should be performed over the entire length of the coding region of the sequence of the present invention (from the start codon to the stop codon, excluding introns). Introns present in other sequences being compared to the sequence of the present invention should also be removed for the pairwise alignment. After aligning the two sequences, in a second step, the identity value is determined based on the generated alignment. The percent identity is calculated as follows: percent identity = (identical residues / length of the alignment region of the sequence of the present invention from the start codon to the stop codon and excluding introns over its entire length) * 100.

[0041] In addition, a preferred nucleic acid sequence alignment program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. [Journal of Molecular Biology] (1979) 48, pp. 443-453) is "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)), using the program default parameters (gap open = 10.0, gap extend = 0.5, and matrix = EDNAFULL).

[0042] Variant polypeptides can also be defined by their sequence similarity when compared to a parental sequence. Sequence similarity is usually provided as "sequence similarity %" or "similarity %". Sequence similarity % takes into account that a defined set of amino acids share similar properties, such as by their size, by their hydrophobicity, by their charge or by other properties. In the present context, the exchange of one amino acid for a similar amino acid can be referred to as a "conservative mutation". The similar amino acids according to the invention are defined as follows, which should also apply to the determination of the similarity % according to the invention, which is also consistent with the BLOSUM62 matrix used, for example, in the program "NEEDLE", which is one of the most commonly used amino acid similarity matrices for database searches and sequence alignments:

[0043] Amino acid A is similar to amino acid S. Amino acid D is similar to amino acids E, N. 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 acid S. Amino acid V is similar to amino acids I, L, M. Amino acid W is similar to amino acids F, Y.

[0044] Amino acid Y is similar to amino acids F, H, W.

[0045] For calculating the sequence similarity, in a first step, a sequence alignment is generated as described above. After aligning two sequences, in a second step, a similarity value is determined based on the generated alignment. For this purpose, the similarity % is calculated as follows: the number of identical residues plus the number of similar residues is divided by the length of the alignment region of the sequences according to the invention over their full length and multiplied by 100: similarity % = [(identical residues + similar residues) / length of the alignment region of the sequences according to the invention over their full length] * 100.

[0046] For nucleic acids, similar sequences can also be determined by hybridization using correspondingly stringent conditions. The term "high stringency conditions" means that a probe of at least 100 nucleotides in length is prehybridized and hybridized at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and 50% formamide, and hybridized for 12 to 24 hours according to standard DNA blotting procedures. Finally, the carrier material is washed three times with 2X SSC, 0.2% SDS at 65°C for 15 minutes each time. The term "very high stringency conditions" means that a probe of at least 100 nucleotides in length is prehybridized and hybridized at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and 50% formamide, and hybridized for 12 to 24 hours according to standard DNA blotting procedures. Finally, the carrier material is washed three times with 2X SSC, 0.2% SDS at 70°C for 15 minutes each time.

[0047] As used herein, "fragment" or "subsequence" refers to a portion of a polynucleotide or amino acid sequence. The term "functional fragment" refers to any nucleic acid or amino acid sequence that contains only a portion of the full-length amino acid sequence but still has the same or similar activity and / or function. 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% identity, 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 fragment contains 100 to 259 contiguous amino acids of the full-length variant polypeptide. Preferably, the functional fragment has at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identity, 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% identity to the original full-length amino acid sequence. Also preferably, the functional fragment retains at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identity, 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 enzymatic activity of the original full-length amino acid sequence. The functional fragment contains contiguous nucleotides or amino acids as compared to the original nucleic acid or original amino acid sequence, respectively. The "original full-length amino acid sequence" is the amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:3 or the variant polypeptide as claimed.

[0048] As used herein, "gene construct" or "expression cassette" is a nucleic acid molecule consisting of at least one sequence of interest to be expressed, which at least one sequence of interest to be expressed is operably linked to one or more control sequences as described herein (linked at least to a promoter).

[0049] As used herein, the term "vector" includes any kind of construct suitable for carrying an exogenous polynucleotide sequence for transfer to another cell or for stable or transient expression within a given cell. As used herein, the term "vector" encompasses any kind of cloning vehicle, such as, but not limited to, plasmids, phagemids, viral vectors (e.g., phage), bacteriophages, baculoviruses, cosmids, fosmids, artificial chromosomes, and any other vector specific for a particular host of interest. The exogenous polynucleotide sequence generally contains a coding sequence that may be referred to herein as a "gene of interest". The gene of interest may contain introns and exons, depending on the type of source or destination of the host cell.

[0050] The term "introduction of polynucleotide" or "transformation of polynucleotide" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for transfer. That is, as used herein, the term "transformation of polynucleotide" is independent of the vector, shuttle system, or host cell, and it encompasses not only transformation by polynucleotide transfer methods known in the art (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), but also any other kind of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0051] A polynucleotide encoding a polypeptide can be "expressed". The term "expression" or "gene expression" means the transcription of a particular gene or genes or a particular nucleic acid construct. The term "expression" or "gene expression" means the transcription of one or more genes or gene constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, followed by translation or non-translation of the latter into protein. The process includes DNA transcription and the processing of the resulting mRNA product.

[0052] The term "purification" or "purifying" refers to a process in which at least one component (e.g., the protein of interest) is separated from at least another component (e.g., particulate matter in the fermentation broth) and transferred to a different compartment or phase, where the different compartment or phase does not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filter membrane or cloth, i.e., the filtrate and the retentate; examples of such different phases are the precipitate and the supernatant or the filter cake and the filtrate. The solution obtained after purifying the enzyme of interest from the fermentation broth is referred to herein as the "purified enzyme solution".

[0053] "Protein formulation" (or "enzyme preparation"), such as "protein variant formulation", means any non-complex formulation containing a small number of components, where these components are for the purpose of stabilizing the protein contained in the protein formulation and / or the stability of the protein formulation itself. Preferably, the non-complex protein formulation contains a higher concentration of protein than the complex formulation (e.g., than a detergent formulation). Thus, preferably, the non-complex protein formulation is a concentrated protein variant formulation. Preferably, the non-complex protein formulation contains 20 to 120 mg / g of active enzyme, while a complex formulation such as a detergent composition contains 0.002 to 10 mg / g of active enzyme.

[0054] "Enzyme properties" include but are not limited to catalytic activity, substrate / cofactor specificity, product specificity, stability over time, thermal stability, pH stability, and chemical stability. "Enzyme activity" or "catalytic activity" means the catalytic effect exerted by an enzyme, expressed as units / mg of enzyme (specific activity) or substrate molecules transformed / minute / enzyme molecule (molecular activity). Enzyme activity can be specified by the actual function of the enzyme. For example, a protease exerts proteolytic activity by catalyzing the hydrolysis cleavage of peptide bonds, a lipase exerts lipolytic activity by the hydrolysis cleavage of ester bonds, amylase activity involves the hydrolysis of glycosidic linkages in polysaccharides, etc. According to the present invention, the enzyme activity is proteolytic activity, which can be determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (Suc-AAPF-pNA; see, for example, DelMar et al. (1979), Analytical Biochem [Analytical Biochemistry] 99, 316-320) or Suc-AAPF-AMC (7-amino-4-methylcoumarin) as a substrate. The cleavage of pNA or AMC from the substrate molecule by proteolytic cleavage results in the release of the yellow color of free pNA or a change in fluorescence properties, which can be quantified by measuring the OD405 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.

[0055] The term "enzyme stability" according to the present invention refers to the retention of enzyme activity as a function of time during storage or operation. The retention of enzyme activity as a function of time during storage is called "storage stability" and is preferred in the context of the present invention.

[0056] To determine and quantify the catalytic activity of an enzyme over time when stored or used under certain conditions, the "initial enzyme activity" is measured at time zero (100%) and at a later time point (x%) under defined conditions. By comparing the measured values, the potential degree of loss of enzyme activity or the degree of residual activity of the enzyme can be determined. The degree of loss of enzyme activity or the degree of residual activity of the enzyme determines the stability or instability of the enzyme.

[0057] As used herein, an "enzyme inhibitor" is a compound that slows down or stops enzyme activity. Enzyme inhibitors generally also stabilize the three-dimensional structure of the enzyme. Thus, enzyme inhibitors generally also act as "enzyme stabilizers".

[0058] "pH stability" refers to the ability of an enzyme to exhibit enzyme activity after exposure to a certain pH value.

[0059] The term "thermal stability (thermalstability, thermostability)" or "temperature-dependent activity" refers to the ability of an enzyme to exhibit catalytic activity or washing performance after exposure to elevated temperatures. Preferably, the enzyme exhibits catalytic activity or washing performance after exposure to a temperature of 40 °C for 14 days (preferably in a detergent composition (preferably, in an ES1-C detergent as described herein)) or after exposure to a temperature of 92 °C for at least 10 minutes.

[0060] The term "detergent stability" or "stability upon storage in a detergent composition" refers to the ability of an enzyme to exhibit catalytic activity or washing performance after storage in a detergent composition (preferably in a detergent composition (preferably, in an ES1-C detergent as described herein) at a temperature of 40 °C or 50 °C for 14 days).

[0061] As used herein, the "washing performance" of an enzyme (also referred to herein as "cleaning performance") refers to the contribution of the enzyme to the cleaning performance of a detergent composition, i.e., the cleaning performance added to the detergent composition by the performance of the enzyme. The term "washing performance" is used herein similarly for laundry washing and hard surface cleaning. Washing performance is compared under relevant washing conditions. The term "relevant washing conditions" is used herein to indicate the conditions actually used in the home in a detergent market segment, in particular washing temperature, time, washing machine, foam concentration, type of detergent, and water hardness. The term "improved washing performance" is used to indicate a better final result in stain removal under relevant washing conditions, or, relative to corresponding control conditions, requiring less enzyme by weight to obtain the same final result.

[0062] As used herein, the term "specific performance" refers to the ability of an enzyme per unit of active enzyme to clean and remove a specific stain or dirt. In some embodiments, the specific performance is determined using stains or dirt such as egg, egg yolk, milk, grass, minced meat, blood, chocolate sauce, baby food, sebum, etc.

[0063] "Detergent composition" or "detergent" means a composition designated for cleaning soiled materials. The detergent compositions according to the present invention include detergent compositions for different applications such as laundry washing and hard surface cleaning. The term "detergent component" is defined herein to mean a class of chemicals that can be used in a detergent composition. A typical detergent component is a surfactant. "Surfactant" (used synonymously herein with "surface active agent") means an organic chemical that, when added to a liquid, changes the properties of that liquid at the interface. Surfactants are referred to as nonionic surfactants, anionic surfactants, cationic surfactants, or amphoteric surfactants according to their ionic charge. The term "effective amount of a detergent component" includes the amount of certain components that provide effective stain removal and / or effective cleaning conditions (such as pH, temperature, water hardness, amount of foaming), the amount that effectively provides optical benefits (such as optical brightening, dye transfer inhibition, color care), and the amount of certain components that effectively assist in processing (maintaining physical properties during processing, storage, and use; such as rheology modifiers, hydrotropes, desiccants). Detergent compositions typically have a protease concentration of 0.002 to 10 mg / g of active enzyme.

[0064] The term "laundry" (or "laundering") relates to both domestic and industrial laundering and means the process of treating textiles and / or fabrics with a solution containing the detergent composition of the present invention. The laundry process can be carried out by using technical devices such as domestic or industrial washing machines. Alternatively, the laundry process can be done manually.

[0065] The term "textile" means any textile material, including yarns (threads made of natural or synthetic fibers for knitting or weaving), yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, and fabrics made from these materials such as clothing, cloth, and other articles. As used herein, the terms "fabric" (a textile made by weaving, knitting, or felting fibers) or "garment" (any piece of clothing made of textile) are also intended to include the broader term textile.

[0066] The term "fiber" includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are fibers of plant origin (such as linen, jute, and cotton) or fibers of animal origin, containing proteins like collagen, keratin, and fibroin (such as silk, sheep's wool, angora wool, mohair, cashmere). Examples of synthetically sourced fibers are polyurethane fibers such as or 、polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. Fibers can be single fibers or parts of textiles, such as knitted fabrics, woven fabrics, or non-woven fabrics.

[0067] The term "hard surface cleaning" relates to both domestic and industrial hard surface cleaning and refers to the process of treating a hard surface with a solution containing the detergent composition of the present invention. The hard surface can include any hard surface in the home or industry, such as floors, furniture, walls, sanitary ceramics, glass, metal surfaces (including knives or tableware), and medical devices such as diagnostic instruments, trays, pans, holders, racks, tweezers, scissors, shears, saws (such as bone saws and their blades), hemostats, knives, chisels, rongeurs, files, pliers, drills, drill bits, rasps, burrs, applicators, lithotripters, elevators, jigs, needle holders, carriers, clamps, hooks, gouges, curettes, retractors, straighteners, punches, extractors, spoons, keratomes, tongue depressors, presses, trocars, dilators, cages, glassware, pipes, catheters, cannulas, plugs, stents, endoscopes, arthroscopes, and related equipment. A particular form of hard surface cleaning is dishwashing, especially automatic dishwashing (ADW).

[0068] The term "washing dishes" refers to all forms of dishwashing, such as manual or automatic dishwashing. Washing dishes includes, but is not limited to, cleaning all forms of pottery, such as plates, cups, glasses, bowls, all forms of cutlery, such as spoons, knives, forks, and serving utensils, and ceramics, plastics such as melamine, metals, porcelain, glass, and acrylics.

[0069] Cleaning performance is evaluated under relevant cleaning conditions. In this context, the term "relevant cleaning conditions" refers to the conditions actually used in a washing machine, an automatic dishwashing machine, or in a manual cleaning process, in particular the cleaning temperature, time, cleaning machinery, foam concentration, detergent type, and water hardness.

[0070] The term "medical device cleaning" refers to the cleaning step of reprocessing reusable medical devices. Medical device cleaning methods can be divided into two categories: manual cleaning methods and mechanical / automatic cleaning methods. Manual cleaning is used when there is no available mechanical device or when the medical device to be cleaned is too fragile or difficult to clean with a mechanical device. Mechanical / automatic cleaning methods remove dirt and microorganisms through an automatic cleaning and rinsing process, which includes ultrasonic cleaning and washing.

[0071] In the field of detergency, the term "stain" is typically used to refer to laundry washing, such as the cleaning of textiles, fabrics, or fibers, while the term "soil" is typically used to refer to hard surface cleaning, such as the cleaning of dishes and cutlery. However, in this document, the terms "stain" and "soil" should be used interchangeably.

[0072] As used herein, "chelating builder" differs from precipitation builder in that when the builder is used in an amount sufficient to combine with all calcium ions in an aqueous solution initially having a hardness of 7 °dH (German hardness) at neutral pH, no substantial precipitation is formed. "Strong builder" is classified as a highly efficient chelating agent that is capable of strongly binding divalent cations (such as Ca2+), and the logarithm of the stability constant (Log K Ca ) of the cation / chelating agent complex is greater than 4, particularly greater than 5, greater than 6 or greater than 7. The stability constant is determined at an ionic strength of 0.1 M and a temperature of 25 °C. "Strong chelating builder" combines the above two characteristics. Strong chelating builders include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), ethylenediamine tetra(methylenephosphonic acid) (EDTMP), nitrilotrimethylphosphonic acid (NTMP), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), methylglycine diacetic acid (MGDA), nitrilotriacetic acid (NTA), 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP), sodium tripolyphosphate (STPP), iminodisuccinic acid (IDS), N,N-diacetic acid tetrasodium salt (GLDA), pyrophosphate, and ethylenediamine disuccinic acid (EDDS).

[0073] "Antimicrobial agent" is a chemical compound that kills microorganisms or inhibits their growth or reproduction. Microorganisms can be bacteria, yeast, or mold. "Preservative" is an antimicrobial agent that can be added to aqueous products and compositions to maintain the original properties, characteristics, and integrity of the products and compositions by killing contaminating microorganisms or inhibiting their growth.

[0074] A composition "substantially free" of a compound shall herein mean that the corresponding compound is not intentionally added to the composition, meaning that there is at most a non-effective amount, and most preferably the composition contains 0% of the compound.

[0075] Detailed description

[0076] In the present invention, novel proteases are provided. More specifically, variants of a parent protease, methods for preparing variant proteases, compositions comprising protease variants, and methods of using variant proteases or compositions comprising these variant proteases are provided.

[0077] Protease variant

[0078] The present invention relates to variant polypeptides having protease activity or fragments of said polypeptides having protease activity, wherein:

[0079] (i) the polypeptide or its fragment has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and

[0080] (ii) Compared with the amino acid sequences shown in SEQ ID NO:1 or SEQ ID NO:3 and referring to the numbering of SEQ ID NO:2, the polypeptide or its fragment contains amino acid substitutions at amino acid residues 43, 78, and 204.

[0081] The variant polypeptide with protease activity of the present invention is a protease that does not occur naturally. Preferably, the variant polypeptide with protease activity of the present invention is a purified, isolated, synthetic, and / or recombinant protease variant. Preferably, the variant polypeptide with protease activity of the present invention is a purified and recombinant protease variant.

[0082] The protease according to the present invention has "proteolytic activity" or "protease activity". "Proteolytic activity" or "protease activity" describes the ability to hydrolyze peptide bonds in polypeptides. Protease activity can be determined by assays known to those skilled in the art for measuring protease activity. Methods for analyzing proteolytic activity are well known in the literature (see, for example, Gupta et al. (2002), Appl. Microbiol. Biotechnol. [Applied Microbiology and Biotechnology] 60: pages 381 - 395). For example, proteolytic activity can be determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA; see, for example, DelMar et al. (1979), Analytical Biochem [Analytical Biochemistry] 99, 316 - 320) or Suc-AAPF-AMC (7-amido-4-methylcoumarin) as substrates. Proteolytic cleavage of pNA or AMC from the substrate molecule results in the release of the yellow color of free pNA or a change in the fluorescence properties, which can be quantified by measuring the OD405 emission at 460 nm after excitation at 380 nm.

[0083] In one embodiment, the variant polypeptide with protease activity of the present invention exhibits 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% of the proteolytic activity of the parental protease (i.e., the protease according to SEQ ID NO:1 or 3, preferably the protease according to SEQ ID NO:3). In a preferred embodiment, the variant polypeptide with protease activity of the present invention exhibits the same proteolytic activity as the parental protease (i.e., the protease according to SEQ ID NO:1 or 3, preferably the protease according to SEQ ID NO:3). More preferably, compared with the parental protease (i.e., the protease according to SEQ ID NO:1 or 3, preferably the protease according to SEQ ID NO:3), the variant polypeptide with protease activity of the present invention exhibits increased proteolytic activity.

[0084] Preferably, the parental protease of the variant polypeptide with protease activity of the present invention is a protease having at least 60% but less than 100% sequence identity with the protease according to SEQ ID NO:1 or 3. Preferably, the parental protease of the variant polypeptide with protease activity of the present invention is the protease according to SEQ ID NO:1 or 3, and more preferably, the parental protease of the variant polypeptide with protease activity of the present invention is the protease according to SEQ ID NO:3. Preferably, the parental protease of the variant polypeptide with protease activity of the present invention is subtilisin (EC 3.4.21.62).

[0085] The present invention relates to a variant polypeptide with protease activity or a fragment of the polypeptide with protease activity, wherein, with reference to the numbering of the amino acid sequence shown in SEQ ID NO:2, the variant polypeptide contains amino acid substitutions at amino acid residues 43, 78 and 204 compared with the parental protease according to SEQ ID NO:1 or 3.

[0086] The parental protease of the variant polypeptide with protease activity of the present invention is a protease having an amino acid sequence with at least 60% sequence identity with SEQ ID NO:1 or 3. Preferably, the parental protease of the variant polypeptide with protease activity of the present invention is the protease according to SEQ ID NO:1 or 3. Most preferably, the parental protease of the variant polypeptide with protease activity of the present invention is the protease according to SEQ ID NO:3.

[0087] In a preferred embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity which, according to the numbering of the amino acid sequence shown in SEQ ID NO:2, compared to the parental sequence, comprises the amino acid substitutions 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 and X204D / E / C / G, preferably wherein the parental protease of the variant polypeptide having protease activity of the present invention is the protease according to SEQ ID NO:1 or 3 or any protease having at least 60% sequence identity with SEQ ID NO:1 or 3, more preferably, the parental protease of the variant polypeptide having protease activity is the protease according to SEQ ID NO:1 or 3, and most preferably, the parental protease of the variant polypeptide having protease activity is the protease according to SEQ ID NO:3.

[0088] In a preferred embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity which, according to the numbering of the amino acid sequence shown in SEQ ID NO:2, compared to the parental sequence, comprises the amino acid substitutions X43K / R, X78N / D and X204D, preferably wherein the parental protease of the variant polypeptide having protease activity of the present invention is the protease according to SEQ ID NO:1 or 3 or any protease having at least 60% sequence identity with SEQ ID NO:1 or 3, more preferably, the parental protease of the variant polypeptide having protease activity is the protease according to SEQ ID NO:1 or 3, and most preferably, the parental protease of the variant polypeptide having protease activity is the protease according to SEQ ID NO:3.

[0089] Preferably, in this embodiment, the amino acid residue at the above-cited position (i.e., X) in the parental protease corresponds to the amino acid residue shown at the corresponding position (according to the numbering of SEQ ID NO:2) in SEQ ID NO:1 or 3.

[0090] In a preferred embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, which, according to the numbering of the amino acid sequence shown in SEQ ID NO:2, contains amino acid substitutions 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 and N204D / E / C / G compared to the parental sequence, preferably wherein the parental protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO:1 or 3 or any protease having at least 60% sequence identity with SEQ ID NO:1 or 3, more preferably, the parental protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO:1 or 3, and most preferably, the parental protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO:3.

[0091] In a preferred embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, which, according to the numbering of the amino acid sequence shown in SEQ ID NO:2, contains amino acid substitutions N43K / R, S78N / D and N204D compared to the parental sequence, preferably wherein the parental protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO:1 or 3 or any protease having at least 60% sequence identity with SEQ ID NO:1 or 3, more preferably, the parental protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO:1 or 3, and most preferably, the parental protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO:3.

[0092] Preferably, the present invention relates to a variant polypeptide having protease activity, which, when compared to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 and with reference to the numbering of the amino acid sequence shown in SEQ ID NO:2, contains amino acid substitutions at amino acid residues N43, S78 and N204.

[0093] Preferably, the present invention relates to a variant polypeptide having protease activity, which, when compared to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 and with reference to the numbering of the amino acid sequence shown in SEQ ID NO:2, contains amino acid substitutions N43K / R, S78N / D and N204D.

[0094] Preferably, the present invention relates to variant polypeptides having protease activity, wherein, with reference to the numbering of the amino acid sequence shown in SEQ ID NO: 2 and compared to the amino acid sequence shown in SEQ ID NO: 3, the variant polypeptide comprises amino acid substitutions at amino acid residues N43, S78 and N204.

[0095] In one embodiment, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide further comprises an amino acid substitution at amino acid residue 76. Preferably, the amino acid substitution at amino acid residue 76 is X76D, and more preferably, the substitution at amino acid residue 76 is N76D.

[0096] Accordingly, the present invention also relates to variant polypeptides having protease activity or fragments of said polypeptides having protease activity, wherein the variant polypeptide comprises the amino acid substitutions 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, X204D / E / C / G and X76D, and wherein the polypeptide or fragment thereof has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0097] Also preferably, the present invention relates to variant polypeptides having protease activity or fragments of said polypeptides having protease activity, wherein the variant polypeptide comprises the amino acid substitutions N43K / R, S78N / D, N204D and N76D, and wherein the polypeptide or fragment thereof has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0098] More preferably, the present invention relates to variant polypeptides having protease activity or fragments of said polypeptides having protease activity, wherein the variant polypeptide comprises the substitutions N43K, S78N, N204D and N76D, and wherein the polypeptide or fragment thereof has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO: 3.

[0099] In one embodiment, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide further comprises an amino acid substitution at amino acid residue 183. Preferably, the amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, more preferably, the substitution at amino acid residue 183 is X183D / E, and even more preferably, the substitution at amino acid residue 183 is N183D.

[0100] Accordingly, the present invention also relates to variant polypeptides having protease activity or fragments of said polypeptides having protease activity, wherein said variant polypeptides comprise one of the following combinations of amino acid substitutions:

[0101] (a) 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, X204D / E / C / G and X183D / E / C / Q / A / M

[0102] (b) 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, X204D / E / C / G, X76D and X183D / E / C / Q / A / M, and

[0103] wherein the polypeptide or fragment thereof has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0104] Preferably, the present invention relates to variant polypeptides having protease activity or fragments of said polypeptides having protease activity, wherein said variant polypeptides comprise one of the following combinations of amino acid substitutions:

[0105] (a) N43K / R, S78N / D, N204D and N183D / E;

[0106] (b) N43K / R, S78N / D, N204D, N76D and N183D / E, and

[0107] wherein the polypeptide or fragment thereof has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0108] More preferably, the present invention relates to variant polypeptides having protease activity or fragments of said polypeptides having protease activity, wherein said variant polypeptides comprise one of the following combinations of amino acid substitutions:

[0109] (a) N43K, S78N, N204D and N183D;

[0110] (b) N43K, S78N, N204D, N76D and N183D, and

[0111] wherein the polypeptide or fragment thereof has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0112] In one embodiment, with reference to the numbering of SEQ ID NO:2, a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, which contains amino acid substitutions at positions 43, 78, and 204 as described above and optionally at positions 76 and / or 183, further contains at least one amino acid substitution at an amino acid residue selected from the group consisting of: 18, 24, 56, 109, 144, 182, 237, 240, 248, 256, and 260.

[0113] In one embodiment, with reference to the numbering of SEQ ID NO:2, a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, which contains amino acid substitutions at positions 43, 78, and 204 as described above and optionally at positions 76 and / or 183, further contains at least one amino acid substitution selected from the group consisting of: X18A / D / C / E / Q, X24K, X56D, X109K / A, X144N / R, X182K / R / E, X237R, X240E / N, X248Q / R, X256E / T / D / R / P, and X260D / K.

[0114] Preferably, with reference to the numbering of SEQ ID NO:2, a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, which contains amino acid substitutions at positions 43, 78, and 204 as described above and optionally at positions 76 and / or 183, further contains at least one amino acid substitution selected from the group consisting of: N18A / D / Q, S24K, S56D, Q109K / A, S144N / R, Q182K / R / E, K237R, S240E / N, N248Q / R, S256E / T / D / R / P, and T260D / K.

[0115] More preferably, with reference to the numbering of SEQ ID NO:2, a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, which contains amino acid substitutions at positions 43, 78, and 204 as described above and optionally at positions 76 and / or 183, further contains at least one amino acid substitution selected from the group consisting of: N18D, S24K, S56D, Q109K, S144N, Q182K, K237R, S240E, N248Q, S256D, and T260D.

[0116] More preferably, with reference to the numbering of SEQ ID NO:2, the variant polypeptide having protease activity or a fragment of said polypeptide having protease activity that contains amino acid substitutions at positions 43, 78 and 204 as described above and optionally at positions 76 and / or 183 further contains at least one amino acid substitution selected from the group consisting of: N18Q, S24K, S56D, Q109K, S144N, Q182K, K237R, S240E, N248Q, S256D and T260D.

[0117] In one embodiment, the variant polypeptide having protease activity or a fragment of said polypeptide having protease activity that contains amino acid substitutions at positions 43, 78 and 204 as described above and optionally at positions 76 and / or 183 does not contain the amino acid substitutions S3T, V4I and V199I.

[0118] In one embodiment, the variant polypeptide having protease activity or a fragment of said polypeptide having protease activity that contains amino acid substitutions at positions 43, 78 and 204 as described above and optionally at positions 76 and / or 183 further contains one of the combinations of amino acid substitutions according to Table 2.

[0119] Preferably, the variant polypeptide having protease activity or a fragment of said polypeptide having protease activity contains the amino acid substitutions 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 and X204D / E / C / G, and further contains one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or its fragment has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0120] Still preferably, the variant polypeptide having protease activity or a fragment of said polypeptide having protease activity contains the amino acid substitutions N43K / R, S78N / D and N204D, and further contains one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or its fragment has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0121] Also preferably, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity comprises the amino acid substitutions N43K, S78N and N204D, and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or its fragment has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0122] Also preferably, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0123] (a) 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, and X204D / E / C / G;

[0124] (b) 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, X204D / E / C / G, and X76D; or

[0125] (c) 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, X204D / E / C / G, and X183D / E / C / Q / A / M;

[0126] (d) 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, X204D / E / C / G, X76D, and X183D / E / C / Q / A / M, and

[0127] further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or its fragment has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0128] Also preferably, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0129] (a) N43K / R, S78N / D, and N204D;

[0130] (b) N43K / R, S78N / D, N204D, and N76D;

[0131] (c) N43K / R, S78N / D, N204D and N183D / E;

[0132] (d) N43K / R, S78N / D, N204D, N76D and N183D / E, and

[0133] further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or a fragment thereof has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0134] Also preferably, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0135] (a) N43K, S78N and N204D;

[0136] (b) N43K, S78N, N204D and N76D;

[0137] (c) N43K, S78N, N204D and N183D;

[0138] (d) N43K, S78N, N204D, N76D and N183D, and

[0139] further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or a fragment thereof has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0140] Also preferably, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein the variant polypeptide comprises the substitutions N183D, N43K, S78N and N204D, and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or a fragment thereof has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:3.

[0141] Also preferably, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein the variant polypeptide comprises the substitutions N183D, N43K, S78N, N204D and N76D, and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or a fragment thereof has an amino acid sequence having at least 60% but less than 100% identity to the amino acid sequence shown in SEQ ID NO:3.

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[0220] Preferably, the variant polypeptide of the present invention comprises a combination of substitutions selected from the group consisting of:

[0221] (a) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q

[0222] (b) X24K; X43K; X78N; X109A; X204D

[0223] (c) X24K; X43K; X78N; X204D; X248R; X260K

[0224] (d) X24K; X43K; X78N; X183D; X204D; X248R; X260K

[0225] (e) X24K; X43K; X78N; X204D; X237R; X248R; X260K

[0226] (f) X24K; X43K; X78N; X204D; X240E; X248R; X260K

[0227] (g) X24K; X43K; X78N; X182E; X183D; X204D; X248R; X260K

[0228] (h) X24K; X43K; X78N; X182E; X204D; X237R; X248R; X260K

[0229] (i) X24K; X43K; X78N; X182E; X204D; X240E; X248R; X260K

[0230] (j) X24K; X43K; X78N; X183D; X204D; X237R; X248R; X260K

[0231] (k) X24K; X43K; X78N; X183D; X204D; X240E; X248R; X260K

[0232] (l) X24K; X43K; X78N; X204D; X237R; X240E; X248R; X260K

[0233] (m) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q

[0234] (n) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q

[0235] (o) X24K; X43K; X78N; X156D; X183D; X204D; X240E; X248R; X260K

[0236] (p) X24K; X43K; X78N; X182E; X183D; X204D; X237R; X248R; X260K

[0237] (q) X24K; X43K; X78N; X182E; X183D; X204D; X240E; X248R; X260K

[0238] (r) X24K; X43K; X78N; X182E; X204D; X237R; X240E; X248R; X260K

[0239] (s) X24K; X43K; X78N; X183D; X204D; X237R; X240E; X248R; X260K

[0240] (t) X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q

[0241] (u) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260K

[0242] (v) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X248Q

[0243] (w) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260D

[0244] (x) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260K

[0245] (y) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X240E; X248Q

[0246] (z)X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260D

[0247] (aa)X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q; X260D(bb)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D(cc)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q(dd)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D(ee)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X240E; X248Q; X260K(ff)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q; X260D(gg)X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R(hh)X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R(ii)X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R, and

[0248] (jj)X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X237A; X240E; X248R.

[0249] Also preferably, variant polypeptides of the invention comprise a combination of substitutions selected from the group consisting of:

[0250] (a)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0251] (b)S24K; N43K; S78N; Q109A; N204D

[0252] (c) S24K; N43K; S78N; N204D; N248R; T260K

[0253] (d) S24K; N43K; S78N; N183D; N204D; N248R; T260K

[0254] (e) S24K; N43K; S78N; N204D; K237R; N248R; T260K

[0255] (f) S24K; N43K; S78N; N204D; S240E; N248R; T260K

[0256] (g) S24K; N43K; S78N; Q182E; N183D; N204D; N248R; T260K

[0257] (h) S24K; N43K; S78N; Q182E; N204D; K237R; N248R; T260K

[0258] (i) S24K; N43K; S78N; Q182E; N204D; S240E; N248R; T260K

[0259] (j) S24K; N43K; S78N; N183D; N204D; K237R; N248R; T260K

[0260] (k) S24K; N43K; S78N; N183D; N204D; S240E; N248R; T260K

[0261] (l) S24K; N43K; S78N; N204D; K237R; S240E; N248R; T260K

[0262] (m) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q

[0263] (n) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0264] (o) S24K; N43K; S78N; S156D; N183D; N204D; S240E; N248R; T260K

[0265] (p) S24K; N43K; S78N; Q182E; N183D; N204D; K237R; N248R; T260K

[0266] (q) S24K; N43K; S78N; Q182E; N183D; N204D; S240E; N248R; T260K

[0267] (r) S24K; N43K; S78N; Q182E; N204D; K237R; S240E; N248R; T260K

[0268] (s) S24K; N43K; S78N; N183D; N204D; K237R; S240E; N248R; T260K

[0269] (t) S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q

[0270] (u) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260K

[0271] (v) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; N248Q

[0272] (w) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260D

[0273] (x) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260K

[0274] (y) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; S240E; N248Q

[0275] (z) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260D

[0276] (aa) S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q; T260D

[0277] (bb)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D

[0278] (cc)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q

[0279] (dd)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D

[0280] (ee)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; S240E; N248Q; T260K(ff)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D(gg)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R;

[0281] (hh)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R;

[0282] (ii)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R, and

[0283] (jj)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R.

[0284] Preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0285] (a)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q

[0286] (b)X24K; X43K; X78N; X109A; X204D

[0287] (c) X24K; X43K; X78N; X204D; X248R; X260K

[0288] (d) X24K; X43K; X78N; X183D; X204D; X248R; X260K

[0289] (e) X24K; X43K; X78N; X204D; X237R; X248R; X260K

[0290] (f) X24K; X43K; X78N; X204D; X240E; X248R; X260K

[0291] (g) X24K; X43K; X78N; X182E; X183D; X204D; X248R; X260K

[0292] (h) X24K; X43K; X78N; X182E; X204D; X237R; X248R; X260K

[0293] (i) X24K; X43K; X78N; X182E; X204D; X240E; X248R; X260K

[0294] (j) X24K; X43K; X78N; X183D; X204D; X237R; X248R; X260K

[0295] (k) X24K; X43K; X78N; X183D; X204D; X240E; X248R; X260K

[0296] (l) X24K; X43K; X78N; X204D; X237R; X240E; X248R; X260K

[0297] (m) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q

[0298] (n) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q

[0299] (o) X24K; X43K; X78N; X156D; X183D; X204D; X240E; X248R; X260K

[0300] (p) X24K; X43K; X78N; X182E; X183D; X204D; X237R; X248R; X260K

[0301] (q) X24K; X43K; X78N; X182E; X183D; X204D; X240E; X248R; X260K

[0302] (r) X24K; X43K; X78N; X182E; X204D; X237R; X240E; X248R; X260K

[0303] (s) X24K; X43K; X78N; X183D; X204D; X237R; X240E; X248R; X260K

[0304] (t) X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q

[0305] (u) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260K

[0306] (v) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X248Q

[0307] (w) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260D

[0308] (x) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260K

[0309] (y) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X240E; X248Q

[0310] (z) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260D

[0311] (aa) X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q; X260D

[0312] (bb)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D

[0313] (cc)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q

[0314] (dd)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D

[0315] (ee)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X240E; X248Q; X260K

[0316] (ff)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q; X260D

[0317] (gg)X18Q; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R

[0318] (hh)X18Q; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R

[0319] (ii)X18Q; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R, and

[0320] (jj)X18Q; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X237A; X240E; X248R.

[0321] Also preferably, variant polypeptides of the invention comprise a combination of substitutions selected from the group consisting of:

[0322] (a)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0323] (b) S24K; N43K; S78N; Q109A; N204D

[0324] (c) S24K; N43K; S78N; N204D; N248R; T260K

[0325] (d) S24K; N43K; S78N; N183D; N204D; N248R; T260K

[0326] (e) S24K; N43K; S78N; N204D; K237R; N248R; T260K

[0327] (f) S24K; N43K; S78N; N204D; S240E; N248R; T260K

[0328] (g) S24K; N43K; S78N; Q182E; N183D; N204D; N248R; T260K

[0329] (h) S24K; N43K; S78N; Q182E; N204D; K237R; N248R; T260K

[0330] (i) S24K; N43K; S78N; Q182E; N204D; S240E; N248R; T260K

[0331] (j) S24K; N43K; S78N; N183D; N204D; K237R; N248R; T260K

[0332] (k) S24K; N43K; S78N; N183D; N204D; S240E; N248R; T260K

[0333] (l) S24K; N43K; S78N; N204D; K237R; S240E; N248R; T260K

[0334] (m) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q

[0335] (n) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0336] (o) S24K; N43K; S78N; S156D; N183D; N204D; S240E; N248R; T260K

[0337] (p) S24K; N43K; S78N; Q182E; N183D; N204D; K237R; N248R; T260K

[0338] (q) S24K; N43K; S78N; Q182E; N183D; N204D; S240E; N248R; T260K

[0339] (r) S24K; N43K; S78N; Q182E; N204D; K237R; S240E; N248R; T260K

[0340] (s) S24K; N43K; S78N; N183D; N204D; K237R; S240E; N248R; T260K

[0341] (t) S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q

[0342] (u) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260K

[0343] (v) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; N248Q

[0344] (w) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260D

[0345] (x) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260K

[0346] (y) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; S240E; N248Q

[0347] (z) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260D

[0348] (aa) S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q; T260D

[0349] (bb)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D

[0350] (cc)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q

[0351] (dd)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D

[0352] (ee)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; S240E; N248Q; T260K

[0353] (ff)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D

[0354] (gg)N18Q; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R;

[0355] (hh)N18Q; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R;

[0356] (ii)N18Q; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R, and

[0357] (jj)N18Q; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R.

[0358] Preferably, the variant polypeptides of the invention comprise a combination of substitutions selected from the group consisting of:

[0359] (a)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q

[0360] (b) X24K; X43K; X78N; X109A; X204D

[0361] (c) X24K; X43K; X78N; X204D; X248R; X260K

[0362] (d) X24K; X43K; X78N; X183D; X204D; X248R; X260K

[0363] (e) X24K; X43K; X78N; X204D; X237R; X248R; X260K

[0364] (f) X24K; X43K; X78N; X204D; X240E; X248R; X260K

[0365] (g) X24K; X43K; X78N; X182E; X183D; X204D; X248R; X260K

[0366] (h) X24K; X43K; X78N; X182E; X204D; X237R; X248R; X260K

[0367] (i) X24K; X43K; X78N; X182E; X204D; X240E; X248R; X260K

[0368] (j) X24K; X43K; X78N; X183D; X204D; X237R; X248R; X260K

[0369] (k) X24K; X43K; X78N; X183D; X204D; X240E; X248R; X260K

[0370] (l) X24K; X43K; X78N; X204D; X237R; X240E; X248R; X260K

[0371] (m) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q

[0372] (n) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q

[0373] (o) X24K; X43K; X78N; X156D; X183D; X204D; X240E; X248R; X260K

[0374] (p) X24K; X43K; X78N; X182E; X183D; X204D; X237R; X248R; X260K

[0375] (q) X24K; X43K; X78N; X182E; X183D; X204D; X240E; X248R; X260K

[0376] (r) X24K; X43K; X78N; X182E; X204D; X237R; X240E; X248R; X260K

[0377] (s) X24K; X43K; X78N; X183D; X204D; X237R; X240E; X248R; X260K

[0378] (t) X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q

[0379] (u) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260K

[0380] (v) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X248Q

[0381] (w) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260D

[0382] (x) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260K

[0383] (y) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X240E; X248Q

[0384] (z) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260D

[0385] (aa) X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q; X260D

[0386] (bb) X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D

[0387] (cc) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q

[0388] (dd) X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D

[0389] (ee) X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X240E; X248Q; X260K

[0390] (ff) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q; X260D

[0391] (gg) X18D; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R

[0392] (hh) X18D; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R

[0393] (ii) X18D; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R, and

[0394] (jj) X18D; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X237A; X240E; X248R.

[0395] Also preferably, variant polypeptides of the invention comprise a combination of substitutions selected from the group consisting of:

[0396] (a) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0397] (b) S24K; N43K; S78N; Q109A; N204D

[0398] (c) S24K; N43K; S78N; N204D; N248R; T260K

[0399] (d) S24K; N43K; S78N; N183D; N204D; N248R; T260K

[0400] (e) S24K; N43K; S78N; N204D; K237R; N248R; T260K

[0401] (f) S24K; N43K; S78N; N204D; S240E; N248R; T260K

[0402] (g) S24K; N43K; S78N; Q182E; N183D; N204D; N248R; T260K

[0403] (h) S24K; N43K; S78N; Q182E; N204D; K237R; N248R; T260K

[0404] (i) S24K; N43K; S78N; Q182E; N204D; S240E; N248R; T260K

[0405] (j) S24K; N43K; S78N; N183D; N204D; K237R; N248R; T260K

[0406] (k) S24K; N43K; S78N; N183D; N204D; S240E; N248R; T260K

[0407] (l) S24K; N43K; S78N; N204D; K237R; S240E; N248R; T260K

[0408] (m) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q

[0409] (n) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0410] (o)S24K; N43K; S78N; S156D; N183D; N204D; S240E; N248R; T260K

[0411] (p)S24K; N43K; S78N; Q182E; N183D; N204D; K237R; N248R; T260K

[0412] (q)S24K; N43K; S78N; Q182E; N183D; N204D; S240E; N248R; T260K

[0413] (r)S24K; N43K; S78N; Q182E; N204D; K237R; S240E; N248R; T260K

[0414] (s)S24K; N43K; S78N; N183D; N204D; K237R; S240E; N248R; T260K

[0415] (t)S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q

[0416] (u)S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260K

[0417] (v)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; N248Q

[0418] (w)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260D

[0419] (x)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260K

[0420] (y)S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; S240E; N248Q

[0421] (z)S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260D

[0422] (aa) S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q; T260D

[0423] (bb) S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D

[0424] (cc) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q

[0425] (dd) S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D

[0426] (ee) S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; S240E; N248Q; T260K

[0427] (ff) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D

[0428] (gg) N18D; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R;

[0429] (hh) N18D; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R;

[0430] (ii) N18D; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R, and

[0431] (jj) N18D; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R.

[0432] Preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0433] (a) X24K; X43K; X56D; X76D; X78N; X144N; X182K; X183D; X204D; X248Q

[0434] (b) X24K; X43K; X76D; X78N; X109A; X204D

[0435] (c) X24K; X43K; X76D; X78N; X204D; X248R; X256D; X260K

[0436] (d) X24K; X43K; X76D; X78N; X183D; X204D; X248R; X256D; X260K

[0437] (e) X24K; X43K; X76D; X78N; X204D; X237R; X248R; X256D; X260K

[0438] (f) X24K; X43K; X76D; X78N; X204D; X240E; X248R; X256D; X260K

[0439] (g) X24K; X43K; X76D; X78N; X182E; X183D; X204D; X248R; X256D; X260K

[0440] (h) X24K; X43K; X76D; X78N; X182E; X204D; X237R; X248R; X256D; X260K

[0441] (i) X24K; X43K; X76D; X78N; X182E; X204D; X240E; X248R; X256D; X260K

[0442] (j) X24K; X43K; X76D; X78N; X183D; X204D; X237R; X248R; X256T; X260K

[0443] (k) X24K; X43K; X76D; X78N; X183D; X204D; X240E; X248R; X256T; X260K

[0444] (l) X24K; X43K; X76D; X78N; X204D; X237R; X240E; X248R; X256D; X260K

[0445] (m) X24K; X43K; X56D; X76D; X78N; X144N; X182E; X183D; X204D; X248Q; X256D

[0446] (n) X24K; X43K; X56D; X76D; X78N; X144N; X182K; X183D; X204D; X248Q; X256D

[0447] (o) X24K; X43K; X76D; X78N; X156D; X183D; X204D; X240E; X248R; X256D; X260K

[0448] (p) X24K; X43K; X76D; X78N; X182E; X183D; X204D; X237R; X248R; X256D; X260K

[0449] (q) X24K; X43K; X76D; X78N; X182E; X183D; X204D; X240E; X248R; X256D; X260K

[0450] (r) X24K; X43K; X76D; X78N; X182E; X204D; X237R; X240E; X248R; X256D; X260K

[0451] (s) X24K; X43K; X76D; X78N; X183D; X204D; X237R; X240E; X248R; X256D; X260K

[0452] (t) X24K; X43K; X56D; X76D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q; X256D

[0453] (u) X24K; X43K; X56D; X76D; X78N; X144N; X182E; X183D; X204D; X248Q; X256D; X260K

[0454] (v) X24K; X43K; X56D; X76D; X78N; X144N; X182K; X183D; X204D; X237R; X248Q; X256D

[0455] (w) X24K; X43K; X56D; X76D; X78N; X144N; X182K; X183D; X204D; X248Q; X256D; X260D

[0456] (x) X24K; X43K; X56D; X76D; X78N; X144N; X182K; X183D; X204D; X248Q; X256D; X260K

[0457] (y) X24K; X43K; X56D; X76D; X78N; X144N; X182E; X183D; X204D; X240E; X248Q; X256D

[0458] (z) X24K; X43K; X56D; X76D; X78N; X144N; X182E; X183D; X204D; X248Q; X256D; X260D

[0459] (aa) X24K; X43K; X56D; X76D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q; X256D; X260D

[0460] (bb) X24K; X43K; X56D; X76D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X256D; X260D

[0461] (cc) X24K; X43K; X56D; X76D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q; X256D

[0462] (dd) X24K; X43K; X56D; X76D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X256D; X260D

[0463] (ee) X24K; X43K; X56D; X76D; X78N; X109K; X144N; X182K; X183D; X204D; X240E; X248Q; X256D; X260K

[0464] (ff) X24K; X43K; X56D; X76D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q; X256D; X260D

[0465] (gg)X18D; X26I; X43K; X56D; X76D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R; X256D

[0466] (hh)X18D; X26I; X43K; X56D; X76D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R; X256D

[0467] (ii)X18D; X26I; X43K; X56D; X76D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R; X256D, and

[0468] (jj)X18D; X26I; X43K; X56D; X76D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X237A; X240E; X248R; X256D.

[0469] Also preferably, variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0470] (a) S24K; N43K; S56D; N76D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0471] (b) S24K; N43K; N76D; S78N; Q109A; N204D

[0472] (c) S24K; N43K; N76D; S78N; N204D; N248R; S256D; T260K

[0473] (d) S24K; N43K; N76D; S78N; N183D; N204D; N248R; S256D; T260K

[0474] (e) S24K; N43K; N76D; S78N; N204D; K237R; N248R; S256D; T260K

[0475] (f) S24K; N43K; N76D; S78N; N204D; S240E; N248R; S256D; T260K

[0476] (g) S24K; N43K; N76D; S78N; Q182E; N183D; N204D; N248R; S256D; T260K

[0477] (h) S24K; N43K; N76D; S78N; Q182E; N204D; K237R; N248R; S256D; T260K

[0478] (i) S24K; N43K; N76D; S78N; Q182E; N204D; S240E; N248R; S256D; T260K

[0479] (j) S24K; N43K; N76D; S78N; N183D; N204D; K237R; N248R; S256T; T260K

[0480] (k) S24K; N43K; N76D; S78N; N183D; N204D; S240E; N248R; S256T; T260K

[0481] (l) S24K; N43K; N76D; S78N; N204D; K237R; S240E; N248R; S256D; T260K

[0482] (m) S24K; N43K; S56D; N76D; S78N; S44N; Q182E; N183D; N204D; N248Q; S256D

[0483] (n) S24K; N43K; S56D; N76D; S78N; S44N; Q182K; N183D; N204D; N248Q; S256D

[0484] (o) S24K; N43K; N76D; S78N; S156D; N183D; N204D; S240E; N248R; S256D; T260K

[0485] (p) S24K; N43K; N76D; S78N; Q182E; N183D; N204D; K237R; N248R; S256D; T260K

[0486] (q) S24K; N43K; N76D; S78N; Q182E; N183D; N204D; S240E; N248R; S256D; T260K

[0487] (r) S24K; N43K; N76D; S78N; Q182E; N204D; K237R; S240E; N248R; S256D; T260K

[0488] (s) S24K; N43K; N76D; S78N; N183D; N204D; K237R; S240E; N248R; S256D; T260K

[0489] (t) S24K; N43K; S56D; N76D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q; S256D

[0490] (u) S24K; N43K; S56D; N76D; S78N; S44N; Q182E; N183D; N204D; N248Q; S256D; T260K

[0491] (v) S24K; N43K; S56D; N76D; S78N; S44N; Q182K; N183D; N204D; K237R; N248Q; S256D

[0492] (w) S24K; N43K; S56D; N76D; S78N; S44N; Q182K; N183D; N204D; N248Q; S256D; T260D

[0493] (x) S24K; N43K; S56D; N76D; S78N; S44N; Q182K; N183D; N204D; N248Q; S256D; T260K

[0494] (y) S24K; N43K; S56D; N76D; S78N; S44N; Q182E; N183D; N204D; S240E; N248Q; S256D

[0495] (z) S24K; N43K; S56D; N76D; S78N; S44N; Q182E; N183D; N204D; N248Q; S256D; T260D

[0496] (aa) S24K; N43K; S56D; N76D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q; S256D; T260D

[0497] (bb)S24K; N43K; S56D; N76D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; S256D; T260D

[0498] (cc)S24K; N43K; S56D; N76D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q; S256D

[0499] (dd)S24K; N43K; S56D; N76D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; S256D; T260D

[0500] (ee)S24K; N43K; S56D; N76D; S78N; Q109K; S44N; Q182K; N183D; N204D; S240E; N248Q; S256D; T260K

[0501] (ff)S24K; N43K; S56D; N76D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q; S256D; T260D

[0502] (gg)N18D; V26I; N43K; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R; S256D

[0503] (hh)N18D; V26I; N43K; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R; S256D

[0504] (ii)N18D; V26I; N43K; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R; S256D, and

[0505] (jj)N18D; V26I; N43K; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R; S256D

[0506] Preferably, compared to SEQ ID NO:1, according to the numbering of SEQ ID NO:2, the variant polypeptide of the present invention contains at least two additional negative charges in the loop region from residue 98 to 104. Preferably, according to the numbering of SEQ ID NO:2, the protease contains the amino acid substitution X101E or X101D. Most preferably, compared to SEQ ID NO:1, according to the numbering of SEQ ID NO:2, the variant polypeptide of the present invention contains the amino acid substitution X101E.

[0507] In an alternative embodiment, according to the numbering of SEQ ID NO:2, the protease variant does not have an amino acid substitution at position 101. Preferably, the protease variant contains the amino acid R101.

[0508] In a preferred embodiment, with reference to the numbering of SEQ ID NO:2, the variant polypeptide of the present invention contains the amino acid residue D or E at position 101. Preferably, it contains E at position 101.

[0509] Preferably, one or more amino acid alterations (preferably substitutions) are at amino acid positions located on the surface of the protease. Preferably, according to the numbering of SEQ ID NO:2, one or more substitutions at amino acid positions located on the surface of the protease are selected from the group consisting of 24, 43, 56, 76, 78, 109, 144, 182, 183, 204, 237, 240, 248, 256, and 260. Preferably, according to the numbering of SEQ ID NO:2, the protease of the present invention contains one or more amino acid substitutions at amino acid positions located on the surface of the protease, wherein the substitutions at amino acid positions located on the surface of the protease are selected from the group consisting of: X24K, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X56D, X76D, 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, X204D / E / C / G, X237R / A, X240E / N, X248Q / R, X256E / T / D / R / P, and X260D / K. Whether an amino acid residue is located on the surface of the protease can be determined by determining the relative accessible surface area or relative solvent accessibility (RSA) of the protein residue, which is a measure of the solvent exposure of the residue. It can be calculated by dividing the ASA (solvent accessible surface area) by the MaxASA (the maximum possible solvent accessible surface area for a given residue). The RSA of surface-exposed amino acids is preferably higher than 0.4, more preferably higher than 0.5 or 0.6.

[0510] Preferably, compared to the parental protease according to SEQ ID NO: 1 or 3, the protease variant comprises 2 to 15 amino acid substitutions. Preferably, compared to the parental protease according to SEQ ID NO: 1 or 3, the protease variant comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 of the above amino acid substitutions.

[0511] The variant polypeptide of the invention comprising the amino acid substitutions described herein and having protease activity preferably has a sequence identity as follows, which has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9% but less than 100% sequence identity with the amino acid sequence of the parental protease (i.e., with the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably with the amino acid sequence according to SEQ ID NO: 3).

[0512] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 12, and preferably, with reference to the numbering of SEQ ID NO: 2, contains amino acid residues 43K, 78N, 183D and 204D.

[0513] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 13, and preferably, with reference to the numbering of SEQ ID NO: 2, contains amino acid residues 43K, 78N and 204D.

[0514] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 14, and preferably, with reference to the numbering of SEQ ID NO: 2, contains amino acid residues 43K, 78N and 204D.

[0515] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 15, and preferably, with reference to the numbering of SEQ ID NO: 2, contains amino acid residues 43K, 78N, 183D and 204D.

[0516] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 16, and preferably, with reference to the numbering of SEQ ID NO: 2, contains amino acid residues 43K, 78N and 204D.

[0517] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 17, and preferably, with reference to the numbering of SEQ ID NO: 2, contains amino acid residues 43K, 78N and 204D.

[0518] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 18, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises the amino acid residues 43K, 78N, 183D and 204D.

[0519] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 19, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises the amino acid residues 43K, 78N and 204D.

[0520] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 20, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises the amino acid residues 43K, 78N and 204D.

[0521] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 21, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises the amino acid residues 43K, 78N, 183D and 204D.

[0522] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 22, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0523] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 23, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N and 204D.

[0524] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 24, and preferably, with reference to the numbering of SEQ ID NO: 2, contains amino acid residues 43K, 78N, 183D and 204D.

[0525] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 25, and preferably, with reference to the numbering of SEQ ID NO: 2, contains amino acid residues 43K, 78N, 183D and 204D.

[0526] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 26, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0527] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 27, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0528] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 28, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0529] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 29, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N and 204D.

[0530] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 30, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0531] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 31, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0532] In one embodiment, the amino acid sequence of the variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 32, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0533] In one embodiment, the amino acid sequence of the variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 33, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0534] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 34, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0535] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 35, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0536] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 36, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises the amino acid residues 43K, 78N, 183D and 204D.

[0537] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 37, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises the amino acid residues 43K, 78N, 183D and 204D.

[0538] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 38, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0539] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 39, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78N, 183D and 204D.

[0540] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 40, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0541] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 41, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0542] In one embodiment, the amino acid sequence of the variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 42, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0543] In one embodiment, the amino acid sequence of the variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 43, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0544] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 44, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78D, 183D and 204D.

[0545] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 45, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78D, 183D and 204D.

[0546] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 46, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78D, 183D and 204D.

[0547] In one embodiment, the amino acid sequence of the variant polypeptide with protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, 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 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identity with the amino acid sequence according to SEQ ID NO: 47, and preferably, with reference to the numbering of SEQ ID NO: 2, contains the amino acid residues 43K, 78D, 183D and 204D.

[0548] The present invention also relates to fragments of the variant polypeptide, wherein the fragment has protease activity. The fragment lacks at least one amino acid as compared to the full-length variant polypeptide. In one embodiment, the fragment of the variant polypeptide comprises 100 to 259 contiguous amino acids of the full-length variant polypeptide. Preferably, the fragment of the variant polypeptide comprises 130 to 259 or 150 to 259 contiguous amino acids of the full-length variant polypeptide. More preferably, the fragment of the variant polypeptide comprises 180 to 259 or 200 to 259 contiguous amino acids of the full-length variant polypeptide. And most preferably, the fragment of the variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259 or 250 to 259 contiguous amino acids of the full-length variant polypeptide.

[0549] In one embodiment, the fragment of the variant polypeptide comprises 100 to 259 contiguous amino acids of the full-length variant polypeptide and has at least 60% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3. Preferably, the fragment of the variant polypeptide comprises 130 to 259 or 150 to 259 contiguous amino acids of the full-length variant polypeptide and has at least 60% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3. More preferably, the fragment of the variant polypeptide comprises 180 to 259 or 200 to 259 contiguous amino acids of the full-length variant polypeptide and has at least 60% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3. And most preferably, the fragment of the variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259 or 250 to 259 contiguous amino acids of the full-length variant polypeptide and has at least 60% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0550] In one embodiment, a fragment of the variant polypeptide comprises 100 to 259 contiguous amino acids of the full-length variant polypeptide and, with reference to the numbering of SEQ ID NO:2, comprises amino acid residues 43, 78, and 204; preferably, the fragment of the variant polypeptide comprises 130 to 259 or 150 to 259 contiguous amino acids of the full-length variant polypeptide and, with reference to the numbering of SEQ ID NO:2, comprises amino acid residues 43, 78, and 204; more preferably, the fragment of the variant polypeptide comprises 180 to 259 or 200 to 259 contiguous amino acids of the full-length variant polypeptide and, with reference to the numbering of SEQ ID NO:2, comprises amino acid residues 43, 78, and 204; and most preferably, the fragment of the variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259, or 250 to 259 contiguous amino acids of the full-length variant polypeptide and, with reference to the numbering of SEQ ID NO:2, comprises amino acid residues 43, 78, and 204.

[0551] In one embodiment, a fragment of the variant polypeptide comprises 100 to 259 contiguous amino acids of the full-length variant polypeptide, has at least 60% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and, with reference to the numbering of SEQ ID NO:2, comprises amino acid residues 43, 78, and 204; preferably, the fragment of the variant polypeptide comprises 130 to 259 or 150 to 259 contiguous amino acids of the full-length variant polypeptide, has at least 60% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and, with reference to the numbering of SEQ ID NO:2, comprises amino acid residues 43, 78, and 204; more preferably, the fragment of the variant polypeptide comprises 180 to 259 or 200 to 259 contiguous amino acids of the full-length variant polypeptide, has at least 60% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and, with reference to the numbering of SEQ ID NO:2, comprises amino acid residues 43, 78, and 204; and most preferably, the fragment of the variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259, or 250 to 259 contiguous amino acids of the full-length variant polypeptide, has at least 60% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and, with reference to the numbering of SEQ ID NO:2, comprises amino acid residues 43, 78, and 204.

[0552] In one embodiment, a fragment of the variant polypeptide retains at least 75%, at least 76%, at least 77%, at least 78%, 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 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 protease activity of the full-length variant polypeptide. The protease activity can be determined as described above.

[0553] The length of the full-length variant polypeptide is 269 amino acids.

[0554] In one embodiment, a fragment of the variant polypeptide comprises 100 to 259 contiguous amino acids of the full-length variant polypeptide according to any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 and 47, preferably, the fragment of the variant polypeptide comprises 130 to 259 or 150 to 259 contiguous amino acids of the full-length variant polypeptide according to any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 and 47, more preferably, the fragment of the variant polypeptide comprises 180 to 259 or 200 to 259 contiguous amino acids of the full-length variant polypeptide according to any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 and 47, and most preferably, the fragment of the variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259 or 250 to 259 contiguous amino acids of the full-length variant polypeptide according to any one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 and 47.

[0555] In one embodiment, compared to the parental protease, preferably compared to the protease according to SEQ ID NO:3, the protease variants of the present invention exhibit one or more improved properties.

[0556] Compared to the parental protease according to SEQ ID NO:3, the variant polypeptide with protease activity of the present invention shows increased stability, especially storage stability. The increased stability is preferably expressed as the residual activity after activation of stability. Preferably, the storage stability is expressed as the residual activity after storage under the corresponding storage conditions, preferably after storage in a detergent composition (preferably in a laundry detergent or dishwashing detergent, preferably in a laundry detergent). Preferably, after storage, the residual activity of the protease variant is increased compared to the residual activity of the parental protease according to SEQ ID NO:3. Preferably, compared to the residual activity of the parental protease, preferably compared to the protease according to SEQ ID NO:3, the residual activity of the protease variant after storage is increased by 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%.

[0557] Preferably, the improved property is one or more properties selected from the group consisting of: increased stability, thermal stability, performance in detergents, performance in laundry detergents, performance in ADW detergents. Preferably, the improved activity is improved washing performance, washing performance of laundry detergents, and / or washing performance of ADW detergents. Preferably, the improved stability is improved thermal stability, thermal stability in a detergent composition (preferably in a laundry detergent composition or a dishwashing detergent composition, preferably in a laundry detergent composition), stability under storage conditions. Preferably, the improved property is improved thermal stability, improved thermal stability in a detergent composition, improved stability under storage in a detergent composition, and / or improved washing performance. Preferably, the improved property is improved thermal stability, preferably, improved thermal stability in a detergent composition, improved stability under storage in a detergent composition, and / or improved washing performance. Preferably, the improved property is improved thermal stability, improved thermal stability in a detergent composition, improved stability under storage conditions, preferably improved stability under storage in a detergent composition, preferably improved stability under storage in a laundry detergent, and / or improved stability under storage in an ADW detergent, improved washing performance, preferably improved washing performance of a laundry detergent and / or improved washing performance of an ADW detergent.

[0558] Preferably, the improved property is improved stability in a detergent composition, preferably a laundry detergent composition.

[0559] Nucleic acid construct

[0560] The present invention also relates to a polynucleotide encoding a 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).

[0561] The present invention also relates to a nucleic acid construct (preferably an expression cassette) comprising the polynucleotide as described herein.

[0562] Typically, an expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region, which in eukaryotes usually contains a polyadenylation site. Additional regulatory elements may include transcriptional as well as translational enhancers. Intron sequences can also be added to the 5' untranslated region (UTR) or the coding sequence to increase the amount of mature messenger accumulated in the cytosol. The expression cassette can be part of a vector or can be integrated into the genome of a host cell and replicated with the genome of its host cell. The expression cassette is generally capable of increasing or decreasing expression.

[0563] The present invention also relates to expression vectors comprising a polynucleotide or nucleic acid construct as described herein. The expression vector can be a low-copy number vector or a high-copy number vector.

[0564] After transformation into a host cell or host cell organelle, the vector as used herein can provide regions for transcription and translation of the foreign polynucleotide. Such additional regions can include regulatory nucleotide sequences, one or more origins of replication required for maintenance and / or replication in a particular cell type, one or more selectable markers, polyadenylation signals, suitable sites for insertion of the foreign coding sequence, such as a multiple cloning site, etc. An example is when the vector needs to be maintained as an episomal genetic element (e.g., a plasmid or cosmid molecule) in a bacterial cell. Non-limiting examples of suitable origins of replication include f1-ori and colE1.

[0565] Without integration into the host cell genome, the vector can replicate, for example, as a plasmid in a bacterial host cell, or it can integrate some or all of its DNA into the host cell genome and thus result in replication and expression of its DNA.

[0566] The polynucleotide encoding the variant polypeptide can be introduced into the vector by standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide comprising the coding sequence can be suitable for introduction (transformation, transduction, transfection, etc.) into a host cell or host cell organelle. A cloning vector suitable for expression of the polynucleotide sequence in the host cell or host cell organelle can be selected.

[0567] Host cell

[0568] The present invention also relates to a host cell comprising a polynucleotide encoding a variant polypeptide of the present invention, a nucleic acid construct as described herein, or an expression vector as described herein. In one embodiment of the present invention, the vector is used for transformation of the host cell.

[0569] The polynucleotide encoding the protease variant as described herein can be introduced into the host cell transiently or stably, and can be maintained non-integrated, for example, as a plasmid. Generally, stable transformation is due to integration of the nucleic acid comprising the foreign coding sequence into the host cell chromosome or as an episome (a separate nuclear DNA fragment). Generally, transient transformation is due to failure to integrate the nucleic acid comprising the foreign nucleic acid sequence into the host cell chromosome or failure to exist as an episome.

[0570] The introduction of nucleic acids into host cells can be achieved, for example but not limited to, by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829 or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751) or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). Specific transformation protocols for various types of host cells are known in the art (see, e.g., for E. coli protoplast transformation, Hanahan, 1983, J. Mol. Biol. 166:557-580).

[0571] A variety of host cells can be used to express the nucleic acid constructs described herein. Host cells containing the nucleic acid constructs described herein can be obtained by one of the methods described herein for introducing polynucleotides into such host cells. The host cells of the invention do not naturally express the protease variant. Thus, the host cells are recombinant host cells; the nucleic acid constructs described herein are heterologous to the host cells.

[0572] In one embodiment, the host cell is a prokaryote or a eukaryote. In another embodiment, the host cell is a bacterium, an archaeon, a fungal cell, a yeast cell or a eukaryotic cell. In another embodiment, the host cell is a non-human host cell.

[0573] In one embodiment, the host cell is a bacterial cell. The bacterial host cell can be any Gram-positive bacterium or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, the genus Bacillus, Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Gram-negative bacteria include, but are not limited to, the genus Escherichia, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Acetobacter, Flavobacterium, Fusobacterium, and Gluconobacter. In a particular embodiment, the bacterial host cell is an Escherichia coli cell. In one embodiment, the host cell is a bacterial cell. In a particular embodiment, the host cell is a cell of the genus Escherichia or Bacillus.

[0574] In the method of the present invention, the bacterial host cell can be any Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus methylotrophicus, Bacillus cereus, Bacillus paralicheniformis, Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the bacterial host cell is a Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In a preferred embodiment, the bacterial host cell is a Bacillus licheniformis cell, Bacillus pumilus, or Bacillus subtilis cell. Preferably, the bacterial host cell is a Bacillus licheniformis cell.

[0575] Preparation method

[0576] Another embodiment of the present invention is a method for obtaining a protease variant of a parental protease, the method comprising the following steps:

[0577] a) Referring to the numbering of the amino acid sequence shown in SEQ ID NO:2, introducing amino acid substitutions at amino acid residues 43, 78, and 204 into the parental protease, preferably into the protease according to SEQ ID NO:1 or SEQ ID NO:3, preferably into the protease according to SEQ ID NO:3, thereby providing a variant polypeptide of the parental protease,

[0578] wherein said variant has at least 60% but less than 100% sequence identity with the amino acid sequence of the polypeptide of SEQ ID NO:1 or 3, preferably SEQ ID NO:3, and wherein said variant polypeptide has protease activity, and preferably wherein said variant polypeptide has improved properties relative to said parental protease.

[0579] The manner of introducing amino acid substitutions into a protein sequence is well known in the art. Any mutagenesis procedure known in the art can be used to prepare variants, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0580] The variant polypeptides obtained by the above procedures can be produced on an industrial scale and subsequently purified. Industrial production of enzymes is typically accomplished by culturing host cells that express the enzyme (also referred to as fermentation). Suitable host cells are described herein. The nucleic acid sequence encoding the variant polypeptides described herein can be transformed into a host cell, and subsequently the host cell can be cultured under conditions suitable for the production of protease variants by the host cell. In a preferred embodiment, the variant polypeptide having protease activity is purified from the host cell.

[0581] Accordingly, in yet another embodiment, the present invention relates to a method for producing a variant polypeptide having protease activity, the method comprising the steps of:

[0582] (a) providing a host cell comprising a heterologous nucleic acid construct containing a polynucleotide encoding a variant polypeptide having protease activity as described herein;

[0583] (b) culturing the recombinant host cell of step (a) under conditions favorable for the expression of the polynucleotide; and

[0584] (c) optionally, recovering the variant polypeptide having protease activity encoded by the polynucleotide.

[0585] Culturing of the host cell is typically carried out in a suitable nutrient medium that permits growth of the recombinant cell and expression of the desired protein. At the end of fermentation, the fermentation broth containing a liquid portion and a solid portion is collected and can be further processed. The variant polypeptide having protease activity can be further purified from the fermentation broth.

[0586] The variant polypeptides with protease activity described herein can be secreted (into the liquid portion of the fermentation broth), or can remain unsecreted from the microbial cells (and thus be contained within the cells of the fermentation broth). Depending on this, protease variants can be recovered from the liquid portion of the fermentation broth or from cell lysates. Preferably, the protease variants are secreted from the cells into the fermentation broth, preferably by adding a secretion signal peptide to the N-terminus of the amino acid sequence, thereby forming a propeptide of the variant polypeptide. After cleavage of the propeptide, the variant polypeptide folds, and the mature protease is released as an active variant. Recovery of the variant polypeptides with protease activity can be achieved by methods known to those skilled in the art. Suitable methods for recovering proteins from fermentation broths include, but are not limited to, collection, centrifugation, filtration, extraction, and precipitation. If the protein of interest precipitates or crystallizes in the fermentation broth or is at least partially bound to the particulate matter of the fermentation broth, additional processing steps may be required to release the protein of interest from the biomass or to dissolve the protein crystals and precipitates. WO 00 / 43502A1, WO 2008 / 110498A1, and WO 2017 / 097869A1 describe methods for recovering a protein of interest from a fermentation broth, which precipitates and / or crystallizes during fermentation. If the desired protein is contained within the cells of the fermentation broth, it may be necessary to release the protein of interest from the cells. Release from the cells can be achieved, for example but not limited to, by cell lysis using techniques well known to those skilled in the art, such as lysozyme treatment, sonication, French press, or a combination thereof.

[0587] The variant polypeptides with protease activity can be purified from the fermentation broth by methods known in the art. For example, protease variants can be isolated from the fermentation broth by conventional procedures, which include but are not limited to centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated polypeptides can then be further purified by a variety of procedures known in the art, which include but are not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). The purified polypeptides can then be concentrated by procedures known in the art, which include but are not limited to ultrafiltration and evaporation, particularly thin-film evaporation.

[0588] Protease composition

[0589] The purification solution of the protease variant described herein can be further processed to form a composition containing the protease. Accordingly, the present invention also claims a composition comprising the protease variant described herein and at least one additional component.

[0590] Thus, the present invention also relates to a method for preparing a composition, the method comprising the steps of: mixing

[0591] i. a protease variant as described herein; and

[0592] ii. one or more components as described herein.

[0593] Furthermore, the present invention also relates to a method for improving the stability of a protease in a composition, the method comprising the steps of: mixing

[0594] (a) a protease variant as described herein; and

[0595] (b) one or more components as described herein.

[0596] The composition can be a non-complex formulation (e.g., a protease variant formulation having one or some other components) or a complex formulation (e.g., a detergent composition).

[0597] In one embodiment of the present invention, the protease variant is formulated as a protease variant formulation, preferably a concentrated protease variant formulation. The protease variant formulation can be solid or liquid. The protein formulation can be obtained by using techniques known in the art. For example, but not limited to, solid enzyme formulations can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described, for example, in WO 94 / 19444A1 and WO 97 / 43482A1.

[0598] The liquid protease variant formulation may contain an enzyme amount in the range of 2% to 40%, 2% to 30%, 2% to 25%, 2% to 12%, or preferably 2%-6% by weight, all relative to the total weight of the enzyme formulation.

[0599] In one embodiment, the protease variant formulation, particularly a liquid enzyme formulation, further comprises one or more additional compounds selected from the group consisting of: solvents, salts, pH regulators, preservatives, enzyme stabilizers, and thickeners. Preferably, the protease variant formulation is substantially free of surfactants, i.e., the protease variant formulation contains less than 1% surfactant, preferably less than 0.5% surfactant. The solvent can be water and / or an organic solvent. The aqueous protease variant formulation of the present invention may contain water in an amount 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 enzyme formulation. The formulation containing the protease variant of the present invention may contain an organic solvent in an amount more than 30% by weight, more than 40% by weight, 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 enzyme formulation. The organic solvent can be a water-miscible solvent. The organic solvent can be one or more selected from the group consisting of glycerol, propylene glycol, polypropylene glycol, and polyethylene glycol.

[0600] In one embodiment, the protease variant formulation contains at least one preservative. Preferably, a preservative means a substance added to a liquid composition for anti-corrosion purposes, meaning more preferably, compounds known to have anti-corrosion characteristics contained in the liquid composition formed during the production process are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of: 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, and formic acid (in acid form or its salt form), and 4,4'-dichlor-2-hydroxy diphenyl ether. Generally, the liquid composition of the present invention contains at least one preservative in an amount less than 10 ppm, such as, relative to the total weight of the liquid composition, the amount ranges from 2 ppm to 5% by weight. Preferably, the protease variant formulation is free of preservatives, which means the content of the preservative is less than 1 ppm, preferably 0 ppm.

[0601] Preferably, the protease variant formulation contains an enzyme stabilization system. Preferably, the enzyme stabilization system contains at least one compound selected from the group consisting of: polyhydric alcohols (preferably, 1,3-propanediol, ethylene glycol, glycerol, 1,2-propanediol, or sorbitol), inorganic salts (preferably, CaCl 2 , MgCl 2 or NaCl), short-chain (preferably, C 1 -C 3)A carboxylic acid or its salt (preferably, formic acid, formate (preferably, sodium formate), acetic acid, acetate, or lactate), borate, boric acid, boronic acid (preferably, 4-formylphenylboronic acid (4-FPBA)), peptide aldehyde (preferably, benzyloxycarbonyl-VAL-H (Z-VAL-H or Cbz-VAL-H) or benzyloxycarbonyl-GAY-H (Z-GAY-H or Cbz-GAY-H)), peptide acetal, and peptide aldehyde bisulfite adduct. Preferably, the enzyme stabilization system comprises a combination of at least two compounds selected from the group consisting of salts, polyols, and short-chain carboxylic acids, and preferably one or more compounds selected from the group consisting of borate, boric acid, boronic acid (preferably, 4-formylphenylboronic acid (4-FPBA)), peptide aldehyde, peptide acetal, and peptide aldehyde bisulfite adduct. In a particularly preferred embodiment, the stabilization system comprises a protease inhibitor, which is preferably selected from borate, boric acid, boronic acid (preferably, 4-FPBA), peptide aldehyde (preferably, peptide aldehyde such as Z-VAL-H or Z-GAY-H), peptide acetal, and peptide aldehyde bisulfite adduct, and preferably the protease inhibitor is a peptide aldehyde, preferably Z-VAL-H or Z-GAY-H. In one embodiment, the stabilization system does not comprise a protease inhibitor. Preferably, the composition is boron-free. Preferably, the protease variant formulation comprises a calcium salt, preferably calcium chloride.

[0602] In one embodiment, the protease variant formulation comprises a protease inhibitor, preferably a peptide aldehyde, in an amount in the range of about 0.05% to 0.8% by weight relative to the total weight of the liquid protease variant formulation. Preferably, the content of the peptide aldehyde is in the range of about 0.1% to 0.6% by weight, about 0.1% to 0.5% by weight, about 0.1% to 0.4% by weight, or about 0.1% to 0.35% by weight, all relative to the total weight of the liquid protease variant formulation.

[0603] Preferably, the liquid protease variant formulation comprises or consists of: a protease variant, a solvent, an enzyme stabilization system, and optionally a preservative and optionally a second enzyme different from the protease variant. Preferably, the protease variant formulation is substantially free of surfactants, i.e., the protease variant formulation comprises less than 1% surfactant, preferably less than 0.5% surfactant.

[0604] Accordingly, the present invention also relates to a method for preparing a protease variant formulation, preferably a concentrated protease variant formulation, the method comprising the step of mixing

[0605] a) a protease variant as described herein; and

[0606] b) one or more components selected from the group consisting of a solvent, an enzyme stabilization system, a preservative, and a second enzyme different from the protease variant.

[0607] Furthermore, the present invention relates to a method for improving the stability of a protease in a composition, the method comprising the step of mixing

[0608] a) a protease variant as described herein; and

[0609] b) one or more components selected from the group consisting of a solvent, an enzyme stabilization system, a preservative, and a second enzyme different from the protease variant.

[0610] In one embodiment, the protease variant described herein is part of a microorganism (live, attenuated or inactivated), a probiotic or a prebiotic.

[0611] Second enzyme

[0612] In another embodiment, a composition comprising a protease variant as described herein further comprises one or more second enzymes different from the protease variant. Preferably, the second enzyme is selected from the group consisting of amylase, one or more proteases other than the protease variant described herein, lipase, cellulase, mannanase, hemicellulase, phospholipase, esterase, pectinase, lactase, peroxidase, xylanase, cutinase, pectate lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanases, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, nuclease, DNase, phosphodiesterase, phytase, carbohydrase, galactanase, xanthanase, xyloglucanase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertase, ribonuclease, transglutaminase, and dispersin, and combinations of at least two of the foregoing types. More preferably, the second enzyme is selected from the group consisting of amylase, lipase, cellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase, and cutinase, and combinations of at least two of the foregoing types. Most preferably, the second enzyme is amylase, preferably α-amylase.

[0613] The composition of the present invention may comprise more than one different type of enzyme (e.g., amylase and protease), or more than one of the same type of enzyme (e.g., two or more different proteases), or a mixture thereof (e.g., amylase and two different proteases, one of the two different proteases being a protease variant as described herein).

[0614] Protease

[0615] Proteases other than the protease variants of the present invention may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with SEQ ID NO:1, with a preference increasing gradually, 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 (numbering according to BPN'), and having proteolytic activity. In one embodiment, such a protease has no mutations at positions Asp32, His64 and Ser221 (numbering referring to SEQ ID NO:2). Preferably, the protease used in combination with the protease variants described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity with SEQ ID NO:1, with a preference increasing gradually, and is further characterized by having an amino acid glutamate (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G) or serine (S) at position 101 (numbering referring to SEQ ID NO:2), preferably glutamate (E), and having proteolytic activity. Most preferably, it is the following protease, which has at least 80% but less than 100% sequence identity with SEQ ID NO:1, and is characterized by having an amino acid glutamate (E) at position 101 (numbering referring to SEQ ID NO:2), and having proteolytic activity. The protease may comprise a single amino acid substitution at position 101 (such as R101E) or a combination of it 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 (numbering referring to SEQ ID NO:2), and having proteolytic activity.In one embodiment, the protease comprises one or more additional substitutions: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine or arginine at position 63 (63A, 63T or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid or glycine at position 217 (217D, 217E or 217G), (i) a combination of two or more amino acids according to (a) to (h), numbered with reference to SEQ ID NO:2. A suitable protease may have at least 80% identity with SEQ ID NO:1 and is characterized by comprising one amino acid (according to (a)-(h)) or a combination according to (i) and amino acid 101E, 101D, 101N, 101Q, 101A, 101G or 101S (numbered with reference to SEQ ID NO:2), and having proteolytic activity. In one embodiment, the protease has at least 80% identity with SEQ ID NO:1 and is characterized by comprising a mutation (numbered with reference to SEQ ID NO:2) R101E, or S3T+V4I+V205I, or S3T+V4I+R101E+V205I or S3T+V4I+V199M+V205I+L217D, and having proteolytic activity. In another embodiment, the protease comprises an amino acid sequence having at least 80% identity with SEQ ID NO:1, and is further characterized by comprising S3T+V4I+S9R+A15T+V68A+D99S+R101S+A103S+I104V+N218D (numbered with reference to SEQ ID NO:2), and having proteolytic activity. In another embodiment, the protease may have an amino acid sequence having at least 80% identity with SEQ ID NO:1, and is further characterized by comprising R101E and one or more substitutions selected from the group consisting of: S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D (numbered with reference to SEQ ID NO:2), and having proteolytic activity.

[0616] Lipase

[0617] "Lipase", "lipolytic enzyme", and "lipid esterase" all refer to enzymes of EC class 3.1.1 ("carboxylic ester hydrolases"). Lipase means an active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; an enzyme having cutinase activity may be referred to herein as cutinase), 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.

[0618] In one aspect of the present invention, suitable lipases (component (b)) are selected from the following: lipases from Humicola (synonym Thermomyces), such as from Humicola lanuginosa (Thermomyces lanuginosus) as described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500, or lipases from Humicola insolens as described in WO 96 / 13580; lipases derived from Rhizomucor miehei as described in WO 92 / 05249; lipases from Pseudomonas strains (some of these are now renamed Burkholderia), for example from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218272, WO 94 / 25578, WO 95 / 30744, WO 95 / 35381, WO 96 / 00292), Pseudomonas cepacia (EP 331376), Pseudomonas stutzeri (GB 1372034), Pseudomonas fluorescens, Pseudomonas sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012), Pseudomonas mendocina (WO 95 / 14783), Pseudomonas glumae (WO 95 / 35381, WO 96 / 00292); lipases from Streptomyces griseus (WO 2011 / 150157) and Streptomyces pristinaespiralis (S.The lipases of (WO2012 / 137147), the GDSL-type Streptomyces lipase (WO 2010 / 065455); the lipase from Thermobifida fusca disclosed in WO 2011 / 084412; the lipase from Geobacillus stearothermophilus disclosed in WO 2011 / 084417; Bacillus lipases, such as those disclosed in WO 00 / 60063, as disclosed by Dartois et al. (1992), Biochemica et Biophysica Acta [Acta Biochemica et Biophysica], 1131, 253 - 360 or the lipase from Bacillus subtilis, Bacillus stearothermophilus (JP S64 - 074992) or Bacillus pumilus (WO 91 / 16422) disclosed in WO 2011 / 084599; the lipase from Candida antarctica disclosed in WO94 / 01541; the cutinase from Pseudomonas mendocina (US 5389536, WO 88 / 09367); the cutinase from Magnaporthe grisea (WO 2010 / 107560); the cutinase from Fusarum solani pisi disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502; and the cutinase from Humicola lanuginosa disclosed in WO 00 / 34450 and WO 01 / 92502.

[0619] Such suitable lipase variants are, for example, those developed by the methods disclosed in WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, WO 00 / 60063, WO 2007 / 087508, EP 407225 and EP 260105.

[0620] Commercially available lipases include, but are not limited to, those sold under the trade names Lipolase TM 、Lipex TM 、Lipolex TM and Lipoclean TMThose sold by Novozymes A / S, Lumafast (originally from Genencor), Preferenz L (DuPont), and Lipomax (Gist-Brocades / now DSM).

[0621] In one embodiment, the lipase is selected from fungal triacylglycerol lipases (EC class 3.1.1.3). The fungal triacylglycerol lipase can be selected from the lipase of Thermomyces lanuginosus. In one embodiment, the Thermomyces lanuginosa lipase is selected from the triacylglycerol lipase of amino acids 1-269 of SEQ ID NO:2 according to US 5869438 and variants thereof having lipolytic activity.

[0622] The Thermomyces lanuginosus lipase can be selected from variants having lipolytic activity that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438.

[0623] The Thermomyces lanuginosus lipase can be selected from variants having lipolytic activity that contain only conservative mutations and do not involve the functional domains of amino acids 1-269 of SEQ ID NO:2 of US 5869438. The lipase variant of this embodiment having lipolytic activity can have at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of US 5869438.

[0624] The Thermomyces lanuginosus lipase can be selected from variants having lipolytic activity that contain the following amino acid substitutions when compared to amino acids 1-269 of SEQ ID NO:2 of US 5869438: T231R and N233R. When compared to amino acids 1-269 of SEQ ID NO:2 of US 5869438, the lipase variant can further contain one or more of the following amino acid exchanges: Q4V, V60S, A150G, L227G, P256K.

[0625] The Thermomyces lanuginosus lipase may be a variant with lipolytic activity that comprises the amino acid substitutions T231R, N233R, Q4V, V60S, A150G, L227G, P256K within the polypeptide sequence of amino acids 1 - 269 of SEQ ID NO:2 of US 5869438 and has at least 95%, at least 96% or at least 97% similarity when compared to the full - length polypeptide sequence of amino acids 1 - 269 of SEQ ID NO:2 of US5869438.

[0626] The Thermomyces lanuginosus lipase may be a variant with lipolytic activity that comprises the amino acid substitutions T231R and N233R within amino acids 1 - 269 of SEQ ID NO:2 of US 5869438 and has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity when compared to the full - length polypeptide sequence of amino acids 1 - 269 of SEQ ID NO:2 of US 5869438.

[0627] The Thermomyces lanuginosus lipase may be a variant with lipolytic activity of amino acids 1 - 269 of SEQ ID NO:2 of US 5869438, wherein the variant of amino acids 1 - 269 of SEQ ID NO:2 of US 5869438 is characterized by containing the amino acid substitutions T231R and N233R.

[0628] The Thermomyces lanuginosus lipase may be selected from the following variants with lipolytic activity that preferably comprise at least one, preferably more than one, more preferably all of the following substitutions within the polypeptide sequence of amino acids 1 - 269 of SEQ ID NO:1 of WO 2015 / 010009: N11K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q, and has at least 95%, at least 96% or at least 97% similarity when compared to the full - length polypeptide sequence of amino acids 1 - 269 of SEQ ID NO:1 of WO 2015 / 010009.

[0629] Amylase

[0630] "Amylase" (α - amylase and / or β - amylase) includes amylases of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, the amylase is selected from the group of α - amylases (EC 3.2.1.1).

[0631] The amylase has "amylolytic activity" or "amylase activity", which involves the (endo)hydrolysis of glycosidic linkages in polysaccharides.

[0632] The amylase can be derived from Bacillus licheniformis having SEQ ID NO:2 as described in WO 95 / 10603 and variants having at least 95% identity therewith. Suitable variants are described in WO 95 / 10603, which contain one or more substitutions at the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444, and which have amylolytic activity. The variants are described in SEQ ID NO:4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424, and WO 99 / 019467.

[0633] The amylase can further be derived from Bacillus stearothermophilus having SEQ ID NO:6 as disclosed in WO 02 / 10355, or an amylase optionally having a C-terminal truncation relative to the wild-type sequence. Suitable variants of SEQ ID NO:6 include variants containing a deletion at position 179 and / or 181 and / or 182 and / or a substitution at position 193.

[0634] The amylase can further be derived from Bacillus sp. 707 having SEQ ID NO:6 as disclosed in WO 99 / 19467 and variants having at least 95% identity therewith. Preferred variants of SEQ NO:6 as disclosed in WO 99 / 19467 are those having a substitution, deletion, or insertion at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269.

[0635] The amylase can further be derived from Bacillus halmapalus having SEQ ID NO:2 or SEQ ID NO:7 as described in WO 96 / 23872, which is also described herein as SP-722. Preferred variants are described in WO 97 / 3296, WO 99 / 194671, and WO 2013 / 001078.

[0636] The amylase can further be derived from Bacillus sp. DSM 12649 having SEQ ID NO:4 as disclosed in WO 00 / 22103 and variants having at least 95% identity therewith.

[0637] The amylase can further be from Bacillus sp. A 7-7 (DSM 12368), which has an amino acid sequence having at least 95% identity with SEQ ID NO:2, particularly with respect to the region of amino acids 32 to 516 according to SEQ ID NO:2, as disclosed in WO 02 / 10356.

[0638] The amylase can further be from Bacillus sp. strain TS-23 having SEQ ID NO:2 and variants thereof as disclosed in WO 2009 / 061380.

[0639] The amylase can further be from a Cytophaga sp. having SEQ ID NO:1 as disclosed in WO 2013 / 184577 and variants having at least 95% identity therewith.

[0640] The amylase can further be from Bacillus megaterium DSM 90 having SEQ ID NO:1 as disclosed in WO 2010 / 104675 and variants having at least 95% identity therewith.

[0641] The amylase can further be from a Bacillus sp. comprising amino acids 1 to 485 of SEQ ID NO:2 as described in WO 00 / 60060 and variants having at least 95% identity therewith.

[0642] The amylase can further be from Bacillus amyloliquefaciens or a variant thereof, preferably selected from the amylases according to SEQ ID NO:3 as described in WO 2016 / 092009.

[0643] The amylase can have the amino acid sequence according to SEQ ID NO:12 as described in WO 2006 / 002643, or can be an amylase variant thereof, which amylase variants comprise the substitutions Y295F and M202LITV within said SEQ ID NO:12.

[0644] The amylase may have the amino acid sequence of SEQ ID NO:6 as described in WO 2011 / 098531, or may be an amylase variant that contains substitutions at one or more positions within SEQ ID NO:6 selected from the group consisting of: 193G,A,S,T,M; 195F,W,Y,L,I,V; 197F,W,Y,L,I,V; 198Q,N; 200F,W,Y,L,I,V; 203F,W,Y,L,I,V; 206F,W,Y,N,L,I,V,H,Q,D,E; 210F,W,Y,L,I,V; 212F,W,Y,L,I,V; 213G,A,S,T,M; and 243F,W,Y,L,I,V.

[0645] The amylase may have the amino acid sequence of SEQ ID NO:1 as described in WO 2013 / 001078, or amylase variants that contain alterations at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 within SEQ ID NO:1.

[0646] The amylase may have the amino acid sequence of SEQ ID NO:2 as described in WO 2013 / 001087, or may be an amylase variant that contains a deletion of positions 181+182, or 182+183, or 183+184 within SEQ ID NO:2, optionally containing one, two, or more modifications at any position corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476, and G477 within SEQ ID NO:2.

[0647] The amylase may be a hybrid α-amylase derived from the above-mentioned amylases, for example, as described in WO 2006 / 066594.

[0648] According to WO 2014 / 183920, a hybrid amylase may have A and B domains and a C domain, where the A and B domains have at least 90% identity with SEQ ID NO:2 of WO 2014 / 183920, and the C domain has at least 90% identity with SEQ ID NO:6 of WO 2014 / 183920, and where the hybrid amylase has amylolytic activity; preferably, the hybrid α-amylase has at least 95% identity with SEQ ID NO:23 of WO 2014 / 183920 and has amylolytic activity.

[0649] According to WO 2014 / 183921, a hybrid amylase may have A and B domains and a C domain, where the A and B domains have at least 75% identity with SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:26, SEQ ID NO:32, and SEQ ID NO:39 as disclosed in WO 2014 / 183921, and the C domain has at least 90% identity with SEQ ID NO:6 of WO 2014 / 183921, and where the hybrid amylase has amylolytic activity; preferably, the hybrid α-amylase has at least 95% identity with SEQ ID NO:30 as disclosed in WO 2014 / 183921 and has amylolytic activity;

[0650] According to WO 2021 / 032881, a hybrid amylase may comprise A and B domains derived from an α-amylase (derived from Bacillus sp. A7-7 (DSM 12368)) and a C domain derived from an α-amylase (from Bacillus cereus); preferably, the A and B domains have at least 75% identity with the amino acid sequence of SEQ ID NO:42, and the C domain has at least 75% identity with the amino acid sequence of SEQ ID NO:44 - both sequences as disclosed in WO 2021 / 032881; more preferably, the hybrid amylase has at least 80% identity with SEQ ID NO:54 as disclosed in WO 2021 / 032881.

[0651] In one embodiment, at least one amylase is selected from commercially available amylases, which include but are not limited to those sold under the trade name Duramyl TM 、Termamyl TM 、Fungamyl TM 、Stainzyme TM 、Stainzyme Plus TM, Natalase TM , Liquozyme X and BAN TM , Amplify TM , Amplify Prime TM (from Novozymes A / S), and Rapidase TM , Purastar TM , Powerase TM , Effectenz TM (M100 from DuPont de Nemours and Company), Preferenz TM (S1000, S110 and F1000; from DuPont de Nemours and Company), PrimaGreen TM (ALL; DuPont de Nemours and Company), Optisize TM (DuPont de Nemours and Company) products sold.

[0652] Mannanase

[0653] As used herein, "mannanase" is an enzyme selected from the group of mannan-degrading enzymes. Mannan-degrading enzymes can be selected from β-mannosidase (EC 3.2.1.25), endo-1,4-β-mannosidase (EC 3.2.1.78), and 1,4-β-mannobiase (EC 3.2.1.100). Preferably, the mannan-degrading enzyme is selected from the group of endo-1,4-β-mannosidase (EC 3.2.1.78), which group of enzymes can be referred to herein as endo-β-1,4-D-mannanase, β-mannanase, or mannanase.

[0654] Mannanase can be selected from the alkaline mannanases of family 5 or 26 (i.e., GH5 or GH26). The term "alkaline mannanase" is intended to cover mannanases having at least 40% of their maximum activity at a given pH of 7 to 12, preferably 7.5 to 10.5.

[0655] The mannanase can be selected from mannanases derived from Bacillus organisms, such as those described below: JP-0304706 [β-mannanase from Bacillus species], JP-63056289 [alkaline, thermostable β-mannanase], JP-63036774 [Bacillus microorganism FERM P-8856 which produces β-mannanase and β-mannosidase at alkaline pH], JP-08051975 [alkaline β-mannanase from alkalophilic Bacillus species AM-001], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 91 / 18974 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 2014 / 100018 [endo-(1,3)-mannanase 1 (Bleman1; see US 5,476,775) cloned from Bacillus circulans or Bacillus lentus strain CMG1240]. Suitable mannanases are described in WO 99 / 064619.

[0656] The mannanase can be selected from mannanases derived from Trichoderma organisms, such as those disclosed in WO 93 / 24622.

[0657] The mannanase can be selected from commercially available mannanases, such as (Novozymes) or (M100) (DuPont).

[0658] Cellulase

[0659] "Cellulase" is an enzyme capable of hydrolyzing cellulose. The cellulase can be selected from cellobiohydrolase (1,4-β-D-glucan cellobiohydrolase, EC 3.2.1.91), endo-β-1,4-glucanase (EC 3.2.1.4), and β-glucosidase (EC 3.2.1.21). The endoglucanase of EC class 3.2.1.4 can be named endoglucanase, endo-1,4-β-D-glucan 4-glucanohydrolase, endo-1,4-β-glucanase, carboxymethylcellulase, and β-1,4-glucanase.

[0660] Endoglucanases can be classified by amino acid sequence similarity (Henrissat, B., accessed 10 / 26 / 2011 at UniProt) in family 5, which contains more than 20 endoglucanases of EC 3.2.1.4. Also refer to T.-M. Enveri, “Microbial Cellulases,” in W.M. Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, pp. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, pp. 200-391 (edited by Wood, W.A. and Kellogg, S.T.); Béguin, P., “Molecular Biology of Cellulose Degradation,” Annu. Rev. Microbiol. (1990), Vol. 44, pp. 219-248; Begun, P. and Aubert, J-P., “The biological degradation of cellulose,” FEMS Microbiology Reviews 13 (1994) pp. 25-58; Henrissat, B., “Cellulases and their interaction with cellulose,” Cellulose (1994), Vol. 1, pp. 169-196.

[0661] Preferably, the cellulase is selected from glycoside hydrolase family 7 (GH7, pfam00840), preferably from endoglucanases (EC 3.2.1.4).

[0662] Preferably, an alkaline cellulase is used, where “alkaline cellulase” is intended to cover cellulases having enzymatic activity at a given pH ranging from 7 to 12, preferably from 7.5 to 10.5.

[0663] In one embodiment, the cellulase is selected from cellulases containing a cellulose-binding domain. In another embodiment, the cellulase contains a catalytic domain but does not contain a cellulose-binding domain.

[0664] In one embodiment, the formulation of the present invention comprises at least one endoglucanase of EC class 3.2.1.4, which is derived from

[0665] · Bacillus, such as Bacillus species CBS 670.93 and CBS 669.93

[0666] · Melanocarpus, such as Melanocarpus albomyces as disclosed in WO 97 / 14804

[0667] · Clostridium, such as Clostridium thermocellum

[0668] · Humicola, such as Humicola insolens (DSM1800) as disclosed in EP 0495257, EP 0531315, EP 0531372, US 4435307, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 94 / 07998 (the sequence of its 43kd human variant), WO 95 / 24471, WO 96 / 11262 and WO 98 / 12307. Figure 1 its 43kd human variant), WO 95 / 24471, WO 96 / 11262 and WO 98 / 12307.

[0669] · Fusarium, such as Fusarium oxysporum, for example, strain J79 (DSM2672) as disclosed in EP0495257, EP0531315, EP 0531372, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO95 / 24471 and WO 96 / 11262

[0670] · Thielavia, such as Thielavia terrestris or Myceliophthora thermophila strain CBS11765 as disclosed in EP 0531315, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 95 / 24471, WO 96 / 11262, WO 96 / 29397 (SEQ ID NO:9 and its variants) and WO 98 / 12307.

[0671] · Trichoderma species, such as Trichoderma reesei, Trichoderma longibrachiatum or Trichoderma harzianum as disclosed in EP 1305432, EP 1240525, WO 92 / 06165, WO 94 / 21801, WO 94 / 26880, WO 95 / 02043, WO 95 / 24471 and WO 02 / 099091.

[0672] · Aspergillus, such as Aspergillus aculeatus as disclosed in WO 93 / 17244.

[0673] · Erwinia, such as Erwinia chrysanthermi as described by M.H. Boyer et al. in European Journal of Biochemistry, Vol. 162, pages 311 - 316 (1987).

[0674] · Acremonium, such as Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, and Acremonium furatum as disclosed in WO 96 / 11262 and WO 96 / 29397 (SEQ ID NO:5 and its variants).

[0675] · Cellvibrio, such as Cellvibrio mixtus DSM11683, Cellvibrio mixtus DSM 11684, Cellvibrio mixtus DSM 11685, Cellvibrio mixtus ACM 2601, Cellvibrio mixtus DSM 1523 and Cellvibrio gilvus DSM 11686 as disclosed in WO 98 / 08940.

[0676] · Cephalosporium, such as Cephalosporium sp. RYM-202 disclosed in WO 96 / 11262.

[0677] Suitable cellulases also include those that are variants of the above cellulases with cellulolytic activity. In one embodiment, the cellulase variant includes a variant having at least 40% to 100% identity when compared to the full-length polypeptide sequence of the parental enzyme disclosed above. In one embodiment, the cellulase variant with cellulolytic activity has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity and / or identity to the full-length polypeptide sequence of the parental enzyme disclosed above.

[0678] The cellulase can be the specific Humicola insolens DSM 1800 cellulase complex having endoglucanase, cellobiohydrolase and β-glucosidase activities.

[0679] The cellulase can be the specific Humicola insolens DSM 1800 endoglucanase (EC 3.2.1.4), preferably having the polypeptide sequence of positions 21 - 435 of SEQ ID NO:2 disclosed in WO 2018 / 224544 or a variant having at least 95% identity thereto.

[0680] The cellulase can be the specific Humicola insolens endoglucanase (EC 3.2.1.4) having 43 kD, preferably according to Figure 1 the polypeptide sequence disclosed in a of WO 94 / 07998 (“43kDhum”) or a variant thereof (preferably having at least 90% identity thereto), preferably those disclosed in WO 94 / 07998.

[0681] The cellulase can be a Bacillus sp. cellulase (EC 3.2.1.4), which is selected from polypeptides having at least 80% similarity and / or identity to the amino acid sequence of positions 1 to 773 of SEQ ID NO:2 of WO 2004 / 053039 or a catalytically active fragment thereof. In one embodiment, the cellulase is a mature polypeptide having at least 95% identity to SEQ ID NO:1 of WO 2018 / 224544.

[0682] The cellulase can be Thielavia terrestris cellulase (EC 3.2.1.4), which has a polypeptide having at least 80% similarity and / or identity with the amino acid sequence from position 1 to position 299 of SEQ ID NO:4 of WO 2004 / 053039 or a catalytically active fragment thereof. In one embodiment, the cellulase is a mature polypeptide having at least 95% identity with SEQ ID NO:4 of WO 2018 / 224544.

[0683] The cellulase can be a mature Sordaria fimicola cellulase, preferably having the polypeptide sequence according to SEQ ID NO:5 of WO 2018 / 224544 or a variant having at least 95% identity therewith.

[0684] The cellulase can be selected from and (Novozymes A / S), Clazinase TM and Puradax HA TM (Genencor Int.Inc.) and KAC-500(B) TM (Kao Corporation).

[0685] Detergent composition

[0686] In one embodiment, the present invention relates to the use of protease variants in detergent compositions. Accordingly, the present invention also relates to a detergent composition comprising a protease variant described herein and one or more detergent components.

[0687] Accordingly, the present invention also relates to a method for preparing a detergent composition, the method comprising the steps of mixing

[0688] a) a protease variant as described herein; and

[0689] b) one or more detergent components as described herein.

[0690] The present invention also relates to a method for preparing a detergent composition having improved protease stability and / or for providing a detergent composition having improved washing performance, the method comprising the steps of mixing

[0691] a) a protease variant as described herein; and

[0692] b) one or more detergent components as described herein.

[0693] The addition of the liquid protease variant formulation to a detergent composition, preferably a liquid detergent composition, is typically carried out at a weight ratio of liquid protease variant formulation:detergent composition of about 1:1000, 1:500, 1:100, 1:50, 1:30, 1:25, 1:20 or 1:10.

[0694] One or more detergent components may optionally be selected from the group consisting of: additional enzymes different from the protease variant, enzyme stabilization systems, surfactants, antifoaming agents, builders, polymers, bleaching systems (bleaching agents), rheology modifiers, hydrotropes, softeners, drying agents, optical brighteners, buffers, preservatives, anti-corrosion additives, dyes and fragrances.

[0695] Preferably, at least one component of the detergent is selected from the group consisting of: surfactants, builders, polymers, preservatives and a second enzyme different from the protease variant. Preferably, one or more detergent components, preferably surfactants and / or builders, are biodegradable and / or bio-based.

[0696] Detergent components may have more than one function in the final application of the detergent composition, and thus any detergent component mentioned in the context of a specific function herein may also have another function in the final application of the detergent composition. The function of a particular detergent component in the final application of the detergent composition generally depends on its amount within the detergent composition, i.e., the effective amount of the detergent component. The type and / or amount of detergent components in the detergent composition vary according to the desired application (such as laundry washing of white textiles, colored textiles and wool). The one or more components selected also depend on the physical form of the detergent composition (liquid, solid, gel, provided in sachets or as tablets, etc.). For example, the selection of one or more components for a laundry washing formulation further depends on regional practices, which are themselves related to aspects such as the washing temperature used, the mechanics of the washing machine (vertical axis compared to horizontal axis machines), the water consumption per wash cycle, etc. and geographical characteristics like the average hardness of the water.

[0697] In one embodiment, the detergent composition is a formulation of more than two detergent components, wherein at least one component is effective in removing stains, at least one component is effective in providing optimal cleaning conditions, and at least one component is effective in maintaining the physical properties of the detergent.

[0698] The detergent composition can be a liquid or solid detergent composition or a combination of liquid and solid detergent compositions. The liquid detergent composition is preferably a gel detergent composition. The solid detergent composition can be a soap bar or a powder detergent composition, preferably a powder detergent composition, wherein the powder detergent composition can be pressed into tablets.

[0699] The detergent composition can be a unit-dose or multi-dose composition. The detergent composition can be in the form of a sachet (including a multi-compartment sachet). The detergent composition can be a laundry or dishwashing detergent composition, suitable for home care and / or industrial and institutional (I&I) cleaning. Both the laundry detergent composition and the dishwashing detergent composition can be in the form of a hand-wash composition or an automatic-wash composition. Preferably, the dishwashing detergent composition is an automatic dishwashing (ADW) composition.

[0700] The detergent sachet can be of any form, shape and material suitable for containing the composition, for example not allowing the composition to be released from the sachet before contact with water. The sachet is made of a water-soluble film that encloses an internal volume. The internal volume can be divided into compartments of the sachet. Preferred films are polymeric materials, preferably polymers forming films or sheets, such as polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). The sachet can contain a solid laundry detergent composition or partial components and / or a liquid detergent composition or partial components separated by a water-soluble film. The compartment of the liquid component can be compositionally different from the compartment containing the solid (see for example US2009 / 0011970).

[0701] In one embodiment, the pH of the detergent composition is in the range of 5 - 12, preferably in the range of 6 - 11, more preferably in a range selected from 6 - 10, 7 - 9 and 7.5 - 8.5. In one embodiment, the formulation is a detergent composition, preferably a liquid detergent composition.

[0702] In one embodiment, the detergent composition according to the present invention comprises one or more surfactants. Surfactants are referred to as non-ionic surfactants, anionic surfactants, cationic surfactants or amphoteric surfactants according to their ionic charge.

[0703] The detergent composition of the present invention may comprise one or more surfactants, and the one or more surfactants may be anionic surfactants and / or cationic surfactants and / or non-ionic surfactants and / or semi-polar surfactants and / or zwitterionic surfactants, or mixtures thereof. In a preferred embodiment, the detergent composition of the present invention comprises at least one surfactant. In a particular embodiment, the detergent composition of the present invention comprises a mixture of one or more non-ionic surfactants and one or more anionic surfactants. The one or more surfactants are typically present at a level of about 0.1 wt.-% to 60 wt.-% (such as 1 wt.-% to 40 wt.-%, 3 wt.-% to 20 wt.-% or 3 wt.-% to 10 wt.-%). The one or more surfactants are selected based on the desired cleaning application and include any one or more conventional surfactants known in the art. Any surfactant known in the art for use in detergents may be used. Non-limiting examples of surfactants are disclosed in McCutcheon's 2016 Detergents and Emulsifiers [McCutcheon's Detergents and Emulsifiers Handbook 2016 Edition], and McCutcheon's 2016 Functional Materials [McCutcheon's Functional Materials Handbook 2016 Edition], both in North American and International editions, MC Publishing Co [MC Publishing Company], 2016 Edition. Other useful examples are disclosed in earlier editions of the same publications known to those skilled in the art.

[0704] When included therein, the detergent typically contains from about 1 wt.-% to 40 wt.-% (such as 5 wt.-% to 30 wt.-%, 5 wt.-% to 15 wt.-% or 20 wt.-% to 25 wt.-%) of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diyl bis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soaps, and combinations thereof.

[0705] When included therein, the detergent typically contains from about 0 to 10 wt.% of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyldimethyl ethanolamine quaternary ammonium salts (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC) and alkylbenzyl dimethylammonium, alkyl quaternary compounds, alkoxylated quaternary (AQA) compounds, and combinations thereof.

[0706] When included therein, the detergent typically contains from about 0.2 wt.-% to 40 wt.-% of a nonionic surfactant, such as from 0.5 wt.-% to 30 wt.-%, particularly from 1 wt.-% to 20 wt.-%, from 3 wt.-% to 10 wt.-%, from 3 wt.-% to 5 wt.-% or from 8 wt.-% to 12 wt.-%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (such as ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides or N-acyl N-alkyl derivatives of glucosamine (glucosamides (GA) or fatty acid glucosamides (FAGA)), and products available under the trade names SPAN and TWEEN, and combinations thereof.

[0707] When included therein, the detergent typically contains from about 0 to 10 wt.-% of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AO), such as alkyldimethylamine oxides, N-(cocoalkyl)-N,N-dimethylamine oxide and N-(tallow-alkyl)-N,N-bis-(2-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0708] When included therein, the detergent typically contains from about 0 to 10 wt.-% of an amphoteric surfactant. Non-limiting examples of amphoteric surfactants include betaines, alkyldimethylbetaines, sulfobetaines, and combinations thereof.

[0709] The detergent composition according to the invention may comprise one or more compounds selected from complexing agents (chelating agents / chelants / sequestrating agents), precipitating agents and ion-exchange compounds, which can form water-soluble complexes with calcium and magnesium. Such compounds may be referred to herein as "builders" or "building reagents", but are not meant to limit such compounds to that function in the final application of the detergent composition.

[0710] In one embodiment, the detergent composition of the present invention comprises at least one builder selected from non-phosphate-based builders, such as sodium gluconate, one or more citrates, one or more silicates, one or more carbonates, one or more phosphonates, one or more aminocarboxylates, one or more polycarboxylates, one or more polysulfonates and one or more polyphosphonates. In one embodiment, the detergent composition of the present invention comprises a strong chelating builder. Preferably, the detergent composition of the present invention is phosphate-free, meaning substantially free of phosphate-based builders. In this context, "substantially phosphate-free" is understood to mean that the sum of the contents of phosphate and polyphosphate is in the range of 10 ppm to 1% by weight, as determined by gravimetric measurement and with reference to the corresponding detergent composition of the present invention. In another preferred embodiment, the detergent composition comprises a phosphonate, wherein the phosphonate is preferably DTPMP and / or HEDP.

[0711] In one embodiment, the detergent composition of the present invention comprises at least one "citrate" selected from monoalkali metal salts and dialkali metal salts of citric acid, in particular monosodium and preferably trisodium citric acid, ammonium or substituted ammonium salts of citric acid and citric acid itself. Citrate can be used as an anhydrous compound or as a hydrate, for example as sodium citrate dihydrate. The total content of citrate can be in the range of 0% to about 20% by weight, in the range of about 0.5% to about 10% by weight or in the range of 1%-5% by weight, all relative to the total weight of the detergent composition. In one embodiment, the detergent composition of the present invention comprises a total amount of citrate in the range of about 1%-3% relative to the total weight of the detergent composition.

[0712] The detergent compositions of the present invention may contain one or more silicates. "One or more silicates" in the context of the present invention include in particular sodium disilicate and sodium metasilicate, aluminosilicates such as sodium aluminosilicates like zeolite A (i.e. Na 12 (AlO 2 ) 12 (SiO 2 ) 12 *27H 2 O) and sheet silicates, in particular those of the formula α-Na 2 Si 2 O 5 , β-Na 2 Si 2 O 5 and δ-Na 2 Si 2 O 5 Those.

[0713] The detergent composition of the present invention may comprise one or more carbonates. The term "one or more carbonates" includes alkali metal carbonates and alkali metal bicarbonates, preferably sodium salts. Particularly suitable is sodium carbonate (Na 2 CO 3 ).

[0714] The detergent composition of the present invention may comprise one or more phosphonates. "Phosphonates" include but are not limited to 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC); ethylenediaminetetra(methylenephosphonic acid) (EDTMPA); 1-hydroxyethane-1,1-diphosphonic acid (HEDP), CH 2 C(OH)[PO(OH) 2 2 ; aminotris(methylenephosphonic acid) (ATMP), N[CH 2 PO(OH) 2 3 ; sodium salt of aminotris(methylenephosphonic acid) (ATMP), N[CH 2 PO(ONa) 2 3 ; 2-hydroxyethyliminobis(methylenephosphonic acid), HOCH 2 CH 2 N[CH 2 PO(OH) 2 2 ; diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), (HO) 2 POCH 2 N[CH 2 CH 2 N[CH 2 PO(OH) 2 2 2 ; sodium salt of diethylenetriaminepenta(methylenephosphonate), C 9 H (28-x) N 3 Na x O 15 P 5 (x = 7); potassium salt of hexamethylenediamine(tetramethylenephosphonic acid), C 10 H (28-x) N 2 K x O 12 P 4 (x = 6); and bis(hexamethylene)triamine(pentamethylenephosphonic acid), (HO 2 )POCH 2 N[(CH 2 ) 2 N[CH 2 PO(OH)​​​​​​2 2 2 Its salts may also be suitable.

[0715] The detergent composition of the present invention may comprise one or more aminocarboxylates. Non-limiting examples of suitable "aminocarboxylates" include, but are not limited to: diethanol glycine (DEG), dimethyl glycine (DMG), nitrilotriacetic acid (NTA), N-hydroxyethyliminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)iminodiacetic acid (HEIDA), hydroxyethylenediaminetriacetic acid, N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA), hydroxyethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid (DTPA), and methylglycine diacetic acid (MGDA), glutamic acid-diacetic acid (GLDA), iminodisuccinic acid (IDS), hydroxyiminodisuccinic acid, ethylenediaminedisuccinic acid (EDDS), aspartic acid-diacetic acid and their alkali metal salts or ammonium salts. Also suitable are aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA) and their alkali metal salts or ammonium salts. Preferred are MGDA or EDDS. As used in this context, the term "ammonium salt" refers to a salt having a cation with at least one nitrogen atom carrying a permanently or temporarily quaternized nitrogen atom. Examples of cations carrying at least one permanently quaternized nitrogen atom include tetramethylammonium, tetraethylammonium, dimethyldiethylammonium, and n-C 10 -C 20 -alkyltrimethylammonium. Examples of cations carrying at least one temporarily quaternized nitrogen atom include protonated amines and ammonia, such as monomethylammonium, dimethylammonium, trimethylammonium, monoethylammonium, diethylammonium, triethylammonium, n-C 10 -C 20 ​​-alkyl dimethylammonium 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, tris(2-hydroxyethyl)ammonium, N-methyl 2-hydroxyethylammonium, N,N-dimethyl-2-hydroxyethylammonium, and especially NH 4 + 。

[0716] In one embodiment, the detergent composition of the present invention comprises more than one builder. Preferably, the detergent composition of the present invention contains less than 0.2% by weight of nitrilotriacetic acid (NTA), or 0.01% to 0.1% by weight of NTA, relative to the total weight of the detergent composition.

[0717] In one embodiment, the detergent composition of the present invention comprises at least one aminocarboxylate selected from methylglycine diacetate (MGDA), glutamic acid diacetate (GLDA), and their corresponding salts, such as their alkali metal (e.g., sodium) salts, in an amount in the range of 0.1% to 25.0% by weight, 1.0% to 18.0% by weight, 3.0% to 15.0% by weight, 3.0% to 10.0% by weight, or 5.0% to 8.0% by weight, relative to the total weight of the detergent composition.

[0718] The detergent composition of the present invention may comprise one or more hydrotropes. The one or more hydrotropes may be selected from organic solvents such as ethanol, isopropanol, ethylene glycol, 1,2-propanediol, and additional organic solvents known in the art that are miscible with water under normal conditions, but are not limited thereto. In one embodiment, the detergent composition of the present invention comprises 1,2-propanediol in an amount in the range of 5% - 10% by weight, preferably about 6% by weight, all relative to the total weight of the detergent composition. Additional non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium 2-ethylhexyl sulfate, and combinations thereof.

[0719] In one embodiment, the detergent composition comprises at least one preservative. Preferably, a preservative means a substance added to the liquid composition for anti-corrosion purposes, meaning more preferably that compounds known to have anti-corrosion characteristics contained in the liquid composition formed during the production process are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of: 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol and formic acid (in acid form or its salt form), and 4,4'-dichlor-2-hydroxy diphenyl ether. Generally, the liquid composition of the present invention comprises at least one preservative in an amount less than 10 ppm, such as in an amount ranging from 2 ppm to 5% by weight relative to the total weight of the liquid composition. Preferably, the liquid composition is free of preservatives, which means the content of the preservative is less than 1 ppm.

[0720] In one embodiment, the detergent composition comprising a protease variant as described herein further comprises one or more second enzymes different from the protease variant. Preferably, the second enzyme is selected from the group consisting of: amylase, a second protease, lipase, cellulase, mannanase, hemicellulase, phospholipase, esterase, pectinase, lactase, peroxidase, xylanase, cutinase, pectate lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, nuclease, DNase, phosphodiesterase, phytase, carbohydrase, galactanase, xanthanase, xyloglucanase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertase, ribonuclease, transglutaminase and dispersin, and combinations of at least two of the foregoing types. More preferably, the second enzyme is selected from the group consisting of: amylase, lipase, cellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase and cutinase, and combinations of at least two of the foregoing types. Most preferably, the second enzyme is amylase, preferably α-amylase.

[0721] Particularly preferred additional enzymes are disclosed elsewhere herein, and this description is also incorporated by reference into this part of the specification.

[0722] The compositions of the present invention may comprise more than one different type of enzyme (such as amylase and protease), or more than one of the same type of enzyme (such as two or more different proteases) or a mixture thereof (such as amylase and two different proteases).

[0723] The detergent composition may comprise a water-soluble calcium and / or magnesium ion source. In one embodiment, the detergent composition comprises an enzyme stabilization system as described herein. Preferably, especially in the case of a liquid detergent composition, the detergent composition may comprise at least one protease inhibitor as described herein, preferably selected from boric acid derivatives (preferably 4-FPBA) and peptide aldehydes (preferably Z-VAL-H or Z-GAY-H). Preferably, the detergent composition is boron-free.

[0724] In one embodiment, the present invention relates to a method for providing a detergent composition, preferably a liquid detergent composition, more preferably a liquid laundry detergent composition, the method comprising the step of mixing in one or more steps:

[0725] (a) at least one protease variant according to the present invention, preferably wherein the protease is provided within a protease variant formulation as described herein; and

[0726] (b) at least one detergent component, the at least one detergent component preferably selected from surfactants, builders, polymers, preservatives, and a second enzyme different from the protease variant, present in an amount effective for the cleaning performance of the detergent and / or for effectively maintaining the physical properties.

[0727] In one embodiment, the present invention relates to a detergent composition comprising

[0728] a) a protease variant as described herein;

[0729] b) one or more surfactants, preferably at a concentration of 0.2% - 65%, preferably 0.2% - 40%,

[0730] c) one or more builders, preferably at a concentration of 0.01% - 25%, and

[0731] d) optionally one or more additional compounds selected from the group consisting of: an additional enzyme different from the protease in a), an antifoaming agent, a polymer, a bleaching system (bleach), a rheology modifier, a hydrotrope, a softener, a desiccant, a brightener, a buffer, a preservative, an anti-corrosion additive, a dye, and a fragrance; preferably wherein the detergent composition is a liquid, powder, sachet or capsule detergent composition.

[0732] Preferably, the detergent composition of the present invention, preferably a powder detergent composition, in addition to comprising a protease variant as described herein, further comprises one or more compounds selected from the group consisting of alcohol ethoxylate 7EO, coconut fatty acid C 12-18 、C 12 -C 14- fatty alcohol ether sulfates (1 - 3 EO, preferably 2 EO), linear alkylbenzene sulfonic acid, sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, HEDP, MGDA, sodium sulfate, sodium chloride, optical brightener, and polymer, and optionally bleach activator and percarbonate.

[0733] Preferably, the detergent composition of the present invention, preferably the powder detergent composition, in addition to containing the protease variant as described herein, further contains

[0734] b) one or more surfactants selected from the group consisting of: alcohol ethoxylates 7 EO, coconut fatty acid C 12-18 , C 12 -C 14 - fatty alcohol ether sulfates (1 - 3 EO, preferably 2 EO), linear alkylbenzene sulfonic acid, preferably at a concentration of 0.2% - 65%,

[0735] c) one or more builders selected from the group consisting of: HEDP, MGDA, GLDA, and DTPMP, preferably at a concentration of 0.01% - 25%, and

[0736] d) one or more compounds selected from the group consisting of: sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, sodium sulfate, sodium chloride, optical brightener, and polymer, and optionally bleach activator and percarbonate.

[0737] Preferably, the detergent composition of the present invention, preferably the liquid detergent composition, in addition to containing the protease variant as described herein, further contains one or more of the compounds selected from the group consisting of: alcohol ethoxylates 7 EO, coconut fatty acid C 12-18 , C 12 -C 14 - fatty alcohol ether sulfates (1 - 3 EO, preferably 2 EO), linear alkylbenzene sulfonic acid, sulfonic acid, 1,2 - propanediol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate, and polymer.

[0738] Preferably, the detergent composition of the present invention, preferably the liquid detergent composition, in addition to containing the protease variant as described herein, further contains

[0739] b) one or more surfactants selected from the group consisting of: alcohol ethoxylates 7 EO, coconut fatty acid C 12-18 , C 12 -C 14 - fatty alcohol ether sulfates (1 - 3 EO, preferably 2 EO), linear alkylbenzene sulfonic acid, preferably at a concentration of 0.2% - 65%,

[0740] c) one or more builders selected from the group consisting of: HEDP, MGDA, GLDA, and DTPMP, preferably at a concentration of 0.01% - 25%, and

[0741] d) one or more compounds selected from the group consisting of: sulfonic acid, 1,2 - propanediol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate, and polymers.

[0742] Preferred formulation

[0743] In one embodiment, the protease variant described herein is included in a formulation comprising one or more, preferably all, compounds selected from the group (all percentages are w / w):

[0744] the protease variant described herein, 0.05% to 0.2%;

[0745] anionic detergent surfactants (such as alkylbenzene sulfonates, alkyl ethoxylated sulfates, and mixtures), 8% to 15%;

[0746] non - ionic detergent surfactants (such as alkyl ethoxylated alcohols), 0.5% to 4%;

[0747] cationic detergent surfactants (such as quaternary ammonium compounds), 0 to 4%;

[0748] other detergent surfactants (such as zwitterionic detergent surfactants, amphoteric surfactants, and mixtures thereof), 0% to 4%;

[0749] carboxylate polymers (such as copolymers of maleic acid and acrylic acid), 1% to 4%;

[0750] polyethylene glycol polymers (such as polyethylene glycol polymers containing polyvinyl acetate side chains), 0.5% to 4%;

[0751] polyester soil - removing polymers (such as Repel - o - tex and / or Texcare polymers), 0.1% to 2%;

[0752] cellulose polymers (such as carboxymethyl cellulose, methyl cellulose, and combinations thereof), 0.5% to 2%;

[0753] other polymers (such as amine polymers, dye transfer inhibitor polymers, hexamethylene diamine derivative polymers, and mixtures thereof), 0% to 4%;

[0754] zeolite builders and phosphate builders (such as zeolite 4A and / or sodium tripolyphosphate), 0% to 4 wt%;

[0755] Other builders (such as sodium citrate and / or citric acid), 0% to 3%;

[0756] Carbonates (such as sodium carbonate and / or sodium bicarbonate), 15% to 30%;

[0757] Silicates (such as sodium silicate), 0% to 10%;

[0758] Fillers (such as sodium sulfate and / or biological fillers), 10% to 40%;

[0759] Available oxygen sources (such as sodium percarbonate), 10% to 20%;

[0760] Bleaching activators (such as tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)), 2% to 8%;

[0761] Bleaching catalysts (such as quaternary oxaziridinium-based bleaching catalysts and / or transition metal bleaching catalysts), 0% to 0.1%;

[0762] Other bleaching agents (such as reducing bleaching agents and / or preformed peracids), 0% to 10%;

[0763] Chelating agents (such as ethylenediamine-N,N'-disuccinic acid (EDDS) and / or hydroxyethane diphosphonic acid (HEDP)), 0.2% to 1%;

[0764] Optical bleaching agents (such as zinc sulfonated phthalocyanine and / or aluminum sulfonated phthalocyanine), 0% to 0.1%;

[0765] Colorants (such as Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof), 0% to 1%;

[0766] Brighteners (such as brightener 15 and / or brightener 49), 0.1% to 0.4%;

[0767] Fabric softeners (such as montmorillonite clay and / or polydimethylsiloxane (PDMS)), 0% to 4%;

[0768] Flocculants (such as polyethylene oxide), 0% to 1%;

[0769] Foam inhibitors (such as silicone and / or fatty acids), 0% to 0.1%;

[0770] Fragrances (such as fragrance microcapsules, spray fragrances, starch-encapsulated fragrance notes, fragrance-loaded zeolites, and any combination thereof), 0.1% to 1%; and

[0771] Cosmetic agents (such as colored soap rings and / or colored spots / stripes), 0% to 1%; and

[0772] Optionally, additional proteases different from the protease variants described herein (e.g., Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, Preferenz P, Progress Uno, Progress Excel, Blaze, Excellenz P), from about 0.05 wt% to about 0.2 wt%;

[0773] Optionally, amylases (e.g., Termamyl(R), Termamyl Ultra(R), Natalase(R), OptisizeHT Plus(R), Purastar, Powerase(R), Stainzyme(R), Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellenz S and any combination thereof), from about 0.05 wt% to about 0.2 wt%;

[0774] Optionally, cellulases (e.g., Carezyme, Celluclean, Puradax, Biotouch, Whitezyme, Revitalenz and combinations thereof), 0.05% to 0.2%;

[0775] Optionally, lipases (e.g., Lipex, Lipolex, Lipoclean, Preferenz L and any combination thereof), 0.05% to 0.2%;

[0776] Optionally, other enzymes (e.g., xyloglucanase, cutinase, pectate lyase (e.g., Xpect), mannanase (e.g., Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F and combinations thereof) bleaching enzymes and combinations thereof), 0.05% to 0.2%;

[0777] The remaining balance.

[0778] In another embodiment, the protease variants described herein are included in a formulation comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w / w):

[0779] The protease variants described herein, 0.05% to 0.2%;

[0780] A polymer containing carboxyl groups (comprising about 60% to about 70% by mass of acrylic-based monomer (A); and about 30% to about 40% by mass of monomer containing sulfonic acid groups (B); and having an average molecular weight of about 23,000 to about 50,000, preferably in the range of about 25,000 to about 38,000 (as described in WO 2014032269)), about 0.5 wt% to about 1.5 wt%;

[0781] Anionic detergent surfactants (such as alkylbenzene sulfonates, alkyl ethoxylated sulfates and mixtures thereof), about 8 wt% to about 15 wt%;

[0782] Nonionic detergent surfactants (such as alkyl ethoxylated alcohols), about 0.5 wt% to 4 wt%;

[0783] Cationic detergent surfactants (such as quaternary ammonium compounds), about 0 wt% to about 4 wt%;

[0784] Other detergent surfactants (such as zwitterionic detergent surfactants, amphoteric surfactants and mixtures thereof), about 0 wt% to 4 wt%;

[0785] Carboxylate polymers (such as copolymers of maleic acid and acrylic acid), about 1 wt% to about 4 wt%;

[0786] Polyethylene glycol polymers (such as polyethylene glycol polymers containing polyvinyl acetate side chains), about 0 wt% to about 4 wt%;

[0787] Polyester soil release polymers (such as Repel-O-Tex(R) and / or Texcare(R) polymers), about 0.1 wt% to about 2 wt%;

[0788] Cellulose polymers (such as carboxymethyl cellulose, methyl cellulose and combinations thereof), about 0.5 wt% to about 2 wt%;

[0789] Other polymers (such as amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers and mixtures thereof), about 0 wt% to about 4 wt%;

[0790] Zeolite builders and phosphate builders (such as zeolite 4A and / or sodium tripolyphosphate), about 0 wt% to about 4 wt%;

[0791] Other builders (such as sodium citrate and / or citric acid), about 0 wt% to about 3 wt%;

[0792] Carbonates (such as sodium carbonate and / or sodium bicarbonate), about 15 wt% to about 30 wt%;

[0793] Silicate (such as sodium silicate), from about 0 wt% to about 10 wt%;

[0794] Filler (such as sodium sulfate and / or biological filler), from about 10 wt% to about 40 wt%;

[0795] Available oxygen source (such as sodium percarbonate), from about 10 wt% to about 20 wt%;

[0796] Bleaching activator (such as tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)), from about 2 wt% to about 8 wt%;

[0797] Bleaching catalyst (such as quaternary oxaziridinium-based bleaching catalyst and / or transition metal bleaching catalyst), from about 0 wt% to about 0.1 wt%;

[0798] Other bleaching agents (such as reducing bleaching agents and / or preformed peracids), from about 0 wt% to about 10 wt%;

[0799] Chelating agent (such as ethylenediamine-N'N'-disuccinic acid (EDDS) and / or hydroxyethane diphosphonic acid (HEDP)), from about 0.2 wt% to about 1 wt%;

[0800] Optical bleaching agent (such as zinc sulfonated phthalocyanine and / or aluminum sulfonated phthalocyanine), from about 0 wt% to about 0.1 wt%;

[0801] Color toner (such as direct violet 99, acid red 52, acid blue 80, direct violet 9, solvent violet 13 and any combination thereof), from about 0 wt% to about 0.5 wt%;

[0802] Brightening agent (such as brightening agent 15 and / or brightening agent 49), from about 0.1 wt% to about 0.4 wt%;

[0803] Fabric softener (such as montmorillonite clay and / or polydimethylsiloxane (PDMS)), from 0 wt% to 15 wt%;

[0804] Flocculant (such as polyethylene oxide), from 0 wt% to 1 wt%;

[0805] Foam inhibitor (such as silicone and / or fatty acid), from 0 wt% to 0.1 wt%;

[0806] Fragrance (such as fragrance microcapsules, spray fragrance, starch-encapsulated fragrance notes, fragrance-loaded zeolite and any combination thereof), from 0.1 wt% to 1 wt%; and

[0807] Cosmetic agent (such as colored soap rings and / or colored spots / strips), from 0 wt% to 1 wt%; and

[0808] Optionally, additional proteases different from the protease variants described herein (such as Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, Preferenz P, Progress Uno, Progress Excel, Blaze, Excellenz P), from about 0.05 wt% to about 0.2 wt%,

[0809] Optionally, amylases (such as Termamyl(R), Termamyl Ultra(R), Natalase(R), OptisizeHT Plus(R), Purastar, Powerase(R), Stainzyme(R), Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellenz S), from about 0.05 wt% to about 0.2 wt%,

[0810] Optionally, cellulases (such as Carezyme(R), Celluzyme(R), Puradax, Celluclean(R), Biotouch, Whitezyme, Revitalenz and combinations thereof, typically having an enzyme activity of about 10 to 50 mg of active enzyme / g), from about 0.05 wt% to 0.5 wt%

[0811] Optionally, lipases (such as Lipex(R), Lipolex(R), Lipoclean(R), Preferenz L and any combination thereof, typically having an enzyme activity of about 10 mg to about 50 mg of active enzyme / g), from about 0.2 wt% to about 1 wt%

[0812] Optionally, other enzymes (such as xyloglucanase (such as Whitezyme(R)), cutinase, pectate lyase (such as Xpect), mannanase (such as Mannanway, Mannastar, Marvellenz, Effectenz M, PreferenzM, Preferenz F and combinations thereof), bleaching enzymes, typically having an enzyme activity of about 10 mg to about 50 mg of active enzyme / g), 0 wt% to 2 wt%,

[0813] The remaining balance.

[0814] Further preferred detergent formulations comprise the components listed below (all percentages are w / w):

[0815] - Water, alcohol ethoxysulfate, alcohol ethoxylate, amine oxide, citric acid, C12-18 capped palm kernel fatty acid, amylase, glycosidase, ethanol, 1,2-propanediol, sodium formate, calcium chloride, sodium hydroxide, silicone emulsion, trans-sulfated EHDQ, protease variant as described herein;

[0816] - Sodium linear alkylbenzene sulfonate 8.8%, ethoxylated fatty alcohol C12-18 (7EO) 4.7%, sodium soap 3.2%, antifoaming agent DC2-4248S 3.9%, sodium aluminosilicate zeolite 4A 28.3%, sodium carbonate 11.6%, sodium salt of copolymer of acrylic acid and maleic acid (Sokalan CP5) 2.4%, sodium silicate 3.0%, carboxymethyl cellulose 1.2%, Dequest 2066 2.8%, optical brightener 0.2%, sodium sulfate 6.5%, amylase 0.4%, protease variant as described herein;

[0817] - 12% LAS, 11% AEO Biosoft N25-7 (NI), 7% AEOS (SLES), 6% MPG (monopropylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soybean soap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1% sodium formate, 0.2% DTM PA and 0.2% PCA, protease variant as described herein;

[0818] - 5%-15% anionic surfactant; <5% nonionic surfactant, phosphonate, soap; enzyme, optical brightener, benzisothiazolinone, methylisothiazolinone, fragrance, α-isomethyl ionone, citronellol, geraniol, linalool, protease variant as described herein;

[0819] - Water, sodium dodecylbenzene sulfonate, C14-C15 alkanol polyether-7, sodium citrate, propylene glycol, sodium palm kernelate, sodium lauryl polyether sulfate, MEA dodecylbenzenesulfonate, quaternized sulfated ethoxylated hexamethylenediamine, sodium cumenesulfonate, fragrance, copolymer of PEG / vinyl acetate, sodium formate, hydrogenated castor oil, diethylenetriamine pentamethylenephosphonic acid sodium salt, PEG / PPG-10 / 2 propylheptyl ether, butylphenyl methylpropional, polyvinylpyridine-N-oxide, sorbitol, glycerol, ethanolamine, sodium hydroxide, α-isomethyl ionone, amylase, calcium chloride, geraniol, linalool, citronellol, tripropylene glycol, glycosidase, benzisothiazolinone, polydimethylsiloxane (Dimethicone), glycosidase, sodium acetate, cellulase, colorant, glyceryl stearate, hydroxyethyl cellulose, silica, protease variant as described herein;

[0820] - Water, sodium lauryl polyether sulfate, propylene glycol, C14-C15 pareth-7, sodium citrate, sodium palmitate, ethanol, sodium formate, quaternized sulfated ethoxylated hexamethylenediamine, sodium hydroxide, fragrance, polyvinylpyridine-N-oxide, sorbitol, calcium chloride, amylase, glycerin, glucosidase, glycosidase, sodium acetate, colorant, cellulase, protease variant as described herein;

[0821] - Water, sodium lauryl polyether sulfate, propylene glycol, C14-C15 pareth-7, sodium citrate, sodium palmitate, ethanol, sodium formate, quaternized sulfated ethoxylated hexamethylenediamine, sodium hydroxide, fragrance, sorbitol, calcium chloride, amylase, glycerin, glucosidase, glycosidase, sodium acetate, colorant, cellulase, protease variant as described herein;

[0822] - Water, sodium lauryl polyether sulfate, propylene glycol, C14-C15 pareth-7, sodium citrate, sodium palmitate, ethanol, sodium formate, quaternized sulfated ethoxylated hexamethylenediamine, sodium hydroxide, sorbitol, calcium chloride, amylase, glycerin, glycosidase, sodium acetate, cellulase, silica, protease variant as described herein;

[0823] - Water, sodium dodecylbenzenesulfonate, C 14 - C 15 pareth-7, sodium citrate, propylene glycol, sodium palmitate, sodium lauryl polyether sulfate, MEA dodecylbenzenesulfonate, quaternized sulfated ethoxylated hexamethylenediamine, sodium cumenesulfonate, fragrance, PEG / vinyl acetate copolymer, sodium formate, C12-C14 pareth-7, hydrogenated castor oil, diethylenetriamine pentamethylenephosphonic acid sodium salt, PEG / PPG-10 / 2 propylheptyl ether, butylphenyl methylpropional, optical brightener, sorbitol, glycerin, ethanolamine, sodium hydroxide, α-isomethyl ionone, amylase, calcium chloride, geraniol, linalool, citronellol, tripropylene glycol, sodium chloride, glycosidase, benzisothiazolinone, polydimethylsiloxane, glycosidase, sodium acetate, cellulase, colorant, glyceryl stearate, hydroxyethyl cellulose, silica, protease variant as described herein;

[0824] - 15%-30% anionic surfactant, non-ionic surfactant, 5%-15% soap, <5% polycarboxylate, fragrance, phosphate, optical brightener, protease variant as described herein;

[0825] - 15%-30% anionic surfactant, 5%-15% non-ionic surfactant, soap, benzisothiazolinone, methylisothiazolinone, fragrance, protease variant as described herein;

[0826] - 11% LAS, 2% AS / AEOS, 2% soap, 3% AEO, 15.15% sodium carbonate, 3% sodium silicate, 18.75% zeolite, 0.15% chelating agent, 2% sodium citrate, 1.65% AA / MA copolymer, 2.5% CMC and 0.5% SRP, protease variant as described herein;

[0827] - 16.5% LAS, 15% zeolite, 12% disodium silicate, 20% sodium carbonate, 1% sokalan, 35.5% sodium sulfate, protease variant as described herein;

[0828] - 15% - 30% anionic surfactant, < 5% non - ionic surfactant, phosphonate, polycarboxylate, zeolite; enzyme, perfume, hexyl cinnamal, protease variant as described herein;

[0829] - 15% - 30% of the following: anionic surfactant, oxygen - based bleaching agent and zeolite, less than 5% of the following: non - ionic surfactant, phosphonate, polycarboxylate, soap, other ingredients: perfume, hexyl cinnamal, benzyl salicylate, linalool, optical brightener, enzyme and citronellol, protease variant as described herein;

[0830] - water, alcohol ethoxysulfate, diethylene glycol, alcohol ethoxylate, ethanolamine, linear alkylbenzene sulfonate, sodium fatty acid, polyethyleneimine ethoxylate, citric acid, sodium cumene sulfonate, propylene glycol, DTPA, disodium diaminodiphenyl ethene disulfonate, dipropylethyltetramine, sodium hydroxide, sodium formate, calcium formate, polydimethylsiloxane, amylase, Liquitint TM , hydrogenated castor oil, perfume, protease variant as described herein;

[0831] - linear alkylbenzene sulfonate, propylene glycol, citric acid, sodium hydroxide, ethanolamine, ethanol, alcohol sulfate, polyethyleneimine ethoxylate, sodium fatty acid, diquaternary ammonium ethoxysulfate, amylase, diethylene glycol, laureth - 9, alkyldimethylamine oxide, perfume, disodium diaminodiphenyl ethene disulfonate, DTPA, sodium formate, calcium formate, polyethylene glycol 4000, mannanase, Liquitint TM Blue, polydimethylsiloxane, protease variant as described herein;

[0832] - Water, sodium laureth sulfate, propylene glycol, ethanol, linear alkylbenzene sulfonate, polyethyleneimine ethoxylate, diethylene glycol, sulfated and ethoxylated hexamethylenediamine, alcohol ethoxylate, MEA salt of linear alkylbenzene sulfonic acid, sodium formate, sodium alkyl sulfate, DTPA, amine oxide, calcium formate, disodium diaminostilbene disulfonate, amylase, polydimethylsiloxane, benzisothiazolinone, protease variant as described herein;

[0833] - Water, alcohol ethoxysulfate, linear alkylbenzene sulfonate, diethylene glycol, propylene glycol, ethanolamine, citric acid, alcohol sulfate, sodium hydroxide, polyethyleneimine ethoxylate, sodium fatty acid, ethanol, amylase, laureth-9, diquaternary ammonium ethoxysulfate, lauramine oxide, sodium cumenesulfonate, fragrance, DTPA, disodium diaminostilbene disulfonate, sodium formate, disodium stilbenyl biphenyl disulfonate, calcium formate, polyethylene glycol 4000, mannanase, pectinase, Liquitint TM Blue, polydimethylsiloxane, protease variant as described herein;

[0834] - Water, sodium laureth sulfate, sodium alkyl sulfate, MEA citrate, MEA salt of linear alkylbenzene sulfonic acid, propylene glycol, diethylene glycol, polyethyleneimine ethoxylate, ethanol, sodium fatty acid, ethanolamine, lauramine oxide, laureth-9, DTPA, sodium cumenesulfonate, sodium formate, calcium formate, linear alkylbenzene sulfonate, alcohol sulfate, sodium hydroxide, diquaternary ammonium ethoxysulfate, fragrance, amylase, mannanase, pectinase, disodium diaminostilbene disulfonate, benzisothiazolinone, Liquitint TM Orange, dipropylethyltetramine, polydimethylsiloxane, cellulase, protease variant as described herein;

[0835] - Water, sodium laureth sulfate, sodium alkyl sulfate, MEA citrate, MEA salt of linear alkylbenzene sulfonic acid, propylene glycol, diethylene glycol, polyethyleneimine ethoxylate, ethanol, sodium fatty acid, ethanolamine, lauramine oxide, laureth-9, DTPA, sodium cumenesulfonate, sodium formate, calcium formate, linear alkylbenzene sulfonate, alcohol sulfate, sodium hydroxide, diquaternary ammonium ethoxysulfate, fragrance, amylase, mannanase, pectinase, disodium diaminostilbene disulfonate, benzisothiazolinone, Liquitint TM Blue, polydimethylsiloxane, dipropylethyltetramine, protease variant as described herein;

[0836] - Water, sodium laureth sulfate, MEA citrate, sodium alkyl sulfate, alcohol ethoxylate, MEA salt of linear alkylbenzene sulfonic acid, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternary ammonium ethoxysulfate, polyethyleneimine, ethoxylated propoxylated compound, ethanol, sodium cumene sulfonate, fragrance, DTPA, disodium diaminostilbene disulfonate, mannanase, cellulase, sodium formate, calcium formate, lauryldimethylamine oxide, Liquitint TM Blue, polydimethylsiloxane / polydimethyl silicone, protease variant as described herein;

[0837] - Water, alcohol ethoxysulfate, linear alkylbenzene sulfonate, alcohol ethoxylate, citric acid, ethanolamine, sodium fatty acid, diethylene glycol, propylene glycol, sodium hydroxide, polyethyleneimine ethoxylate, silicone polyether, ethanol, amylase, sodium cumene sulfonate, diquaternary ammonium ethoxysulfate, laureth-9, fragrance, DTPA, disodium diaminostilbene disulfonate, disodium stilbenyl biphenyl disulfonate, sodium formate, calcium formate, mannanase, Liquitint TM Orange, polydimethylsiloxane, polyacrylamide quaternary ammonium chloride, cellulase, dipropylethyltetramine, protease variant as described herein;

[0838] - Water, alcohol ethoxysulfate, diethylene glycol, monoethanolamine citrate, sodium formate, propylene glycol, linear alkylbenzene sulfonate, ethanolamine, ethanol, polyethyleneimine ethoxylate, amylase, benzisothiazolinone, calcium formate, citric acid, sodium diethylenetriaminepentaacetate, polydimethylsiloxane, diquaternary ammonium ethoxysulfate, disodium diaminostilbene disulfonate, laureth-9, mannanase, sodium cumene sulfonate, sodium fatty acid, protease variant as described herein;

[0839] - Water, sodium laureth sulfate, MEA citrate, alcohol sulfate, alcohol ethoxylate, MEA salt of linear alkylbenzene sulfonic acid, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternary ammonium ethoxysulfate, ethoxylated propoxylated polyethyleneimine ethoxylate, ethanol, sodium cumene sulfonate, fragrance, DTPA, disodium diaminostilbene disulfonate, mannanase, cellulase, amylase, sodium formate, calcium formate, lauryldimethylamine oxide, Liquitint TM Blue, polydimethylsiloxane, protease variant as described herein;

[0840] - Water, sodium laureth sulfate, MEA citrate, linear alkylbenzene sulfonate: sodium salt, alcohol ethoxylate, linear alkylbenzene sulfonate: MEA salt, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternary ammonium ethoxysulfate, amylase, polyethyleneimine ethoxylate propoxylate, ethanol, sodium cumenesulfonate, citric acid, DTPA, disodium diaminostilbene disulfonate, sodium formate, calcium formate, polydimethylsiloxane, protease variant as described herein;

[0841] - Water, alcohol ethoxylate sulfate, sodium linear alkylbenzene sulfonate / Mea salt, propylene glycol, diethylene glycol, sodium formate, ethanol, sodium fatty acid, fragrance, lauryl amine oxide, DTPA, polyethyleneamine ethoxylate, calcium formate, disodium diaminostilbene disulfonate, polydimethylsiloxane, tetraamine, Liquitint TM Blue, protease variant as described herein;

[0842] - Linear alkylbenzene sulfonate, C 12-16 Chain alkanol polyether - 9, propylene glycol, alcohol ethoxysulfate, water, polyethyleneimine ethoxylate, glycerol, fatty acid salt, PEG - 136 polyvinyl acetate, ethylenediamine disuccinate, citric acid monoethanolamine, sodium bisulfite, sodium diethylenetriaminepentaacetate, disodium stilbenyl biphenyl disulfonate, calcium formate, mannanase, xyloglucanase, sodium formate, hydrogenated castor oil, natalase, dye, termamyl, subtilisin, benzisothiazoline, fragrance, protease variant as described herein;

[0843] - Deionized water, dipropylene glycol butyl ether, sodium alkyl sulfate, hydrogen peroxide, ethanol, magnesium sulfate, alkyl dimethyl amine oxide, citric acid, sodium hydroxide, trimethoxybenzoic acid, fragrance, protease variant as described herein;

[0844] - Water, alkyl ethoxylate, linear alkylbenzene sulfonate, hydrogen peroxide, diquaternary ammonium ethoxysulfate, ethanolamine, disodium stilbenyl biphenyl disulfonate, tetrabutylethylene bisphenol, F&DC Yellow 3, fragrance, protease variant as described herein;

[0845] - Sodium percarbonate, sodium sulfate, sodium carbonate, sodium aluminosilicate, nonanoyloxybenzenesulfonate, sodium polyacrylate, water, sodium alkylbenzene sulfonate, DTPA, polyethylene glycol, sodium palmitate, amylase, modified starch, FD&C Blue 1, fragrance, protease variant as described herein;

[0846] - Water, alkyl ethoxylate, MEA borate, linear alkylbenzene sulfonate, propylene glycol, diquaternary ammonium ethoxysulfate, Calcium Chloride enzyme, ethanolamine, benzisothiazolinone, amylase, sodium citrate, sodium hydroxide, fragrance, protease variant as described herein;

[0847] - Water, amine oxide, dipropylene glycol phenyl ether, hydrogen peroxide, citric acid, ethylenediamine disuccinate, sodium lauryl sulfate, fragrance, amylase having at least 91% sequence identity with SEQ ID NO:1, protease variant as described herein;

[0848] - Sodium bicarbonate, sodium carbonate, sodium percarbonate, alcohol ethoxylate, sodium chloride, maleic / acrylic acid copolymer, nonanoyloxybenzenesulfonate, sodium sulfate, colorant, sodium diethylenetriaminepentaacetate, hydrated aluminosilicate (zeolite), polyethylene glycol, sodium alkylbenzene sulfonate, sodium palmitate, starch, water, fragrance, protease variant as described herein;

[0849] - Polyvinyl alcohol bag film, which contains a liquid part and a powder part: Liquid components: dipropylene glycol, diquaternary ammonium ethoxysulfate, water, glycerol, Liquitint TM Orange, protease variant as described herein;

[0850] - Powder components: sodium percarbonate, nonanoyloxybenzenesulfonate, sodium carbonate, sodium sulfate, sodium aluminosilicate, sodium polyacrylate, sodium alkylbenzene sulfonate, maleic / acrylic acid copolymer, water, amylase, polyethylene glycol, sodium palmitate, modified starch, glycerol, DTPA, fragrance, protease variant as described herein;

[0851] - Water, sodium lauryl alcohol ethoxysulfate, linear alkylbenzene sulfonate / MEA salt, MEA citrate, propylene glycol, polyethyleneimine ethoxylate, ethanol, diethylene glycol, polyethyleneimine propoxyethoxylate, sodium fatty acid, sodium cumene sulfonate, DTPA, fragrance, amylase, disodium diaminostilbene disulfonate, calcium formate, sodium formate, glucoamylase, polydimethylsiloxane, Liquitint TM Blue, mannanase, protease variant as described herein;

[0852] - Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, bentonite, water, sodium percarbonate, sodium polyacrylate, silicate, alkyl sulfate, nonanoyloxybenzenesulfonate, DTPA, polyethylene glycol 4000, silicone, ethoxylate, fragrance, polyethylene oxide, palmitic acid, disodium diaminostilbene disulfonate, amylase, Liquitint TM Red, FD&C Blue1, cellulase, protease variant as described herein;

[0853] - Water, sodium laureth sulfate, MEA citrate, linear alkylbenzene sulfonate: sodium / MEA salt, propylene glycol, polyethyleneimine ethoxylate, ethanol, diethylene glycol, polyethyleneimine, propoxylated ethoxylate, diquaternary ammonium ethoxysulfate, alcohol sulfate, polydimethylsiloxane, fragrance, sodium fatty acid, DTPA, sodium bisulfite, disodium diaminostilbene disulfonate, amylase, glucoamylase, castor oil, calcium formate, MEA, styrene acrylic copolymer, sodium formate, Liquitint TM Blue, protease variants as described herein;

[0854] - Water, sodium laureth sulfate, MEA citrate, linear alkylbenzene sulfonate: sodium / MEA salt, propylene glycol, ethanol, diethylene glycol, polyethyleneimine propoxylated ethoxylate, polyethyleneimine ethoxylate, alcohol sulfate, polydimethylsiloxane, fragrance, sodium fatty acid, DTPA, amylase, sodium bisulfite, disodium diaminostilbene disulfonate, castor oil, calcium formate, MEA, styrene acrylic copolymer, propylammonium propionamide, glucoamylase, sodium formate, Liquitint TM Blue, protease variants as described herein;

[0855] - Water, sodium laureth sulfate, MEA citrate, linear alkylbenzene sulfonate: sodium / MEA salt, propylene glycol, polyethyleneimine ethoxylate, ethanol, diethylene glycol, polyethyleneimine propoxylated ethoxylate, diquaternary ammonium ethoxysulfate, alcohol sulfate, polydimethylsiloxane, fragrance, sodium fatty acid, DTPA, sodium bisulfite, disodium diaminostilbene disulfonate, amylase, glucoamylase, castor oil, calcium formate, MEA, styrene acrylic copolymer, propylammonium propionamide, sodium formate, Liquitint TM Blue, protease variants as described herein;

[0856] - Sodium carbonate, sodium aluminosilicate, alkyl sulfate, sodium sulfate, linear alkylbenzene sulfonate, water, sodium polyacrylate, silicate, ethoxylate, sodium percarbonate, polyethylene glycol 4000, amylase, disodium diaminostilbene disulfonate, silicone, cellulase, protease variants as described herein;

[0857] - Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, alkyl sulfate, sodium percarbonate, water, sodium polyacrylate, silicate, nonanoyloxybenzenesulfonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, disodium diaminostilbene disulfonate, palmitic acid, amylase, silicone, cellulase, protease variants as described herein;

[0858] - Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, water, nonanoyloxybenzenesulfonate, alkyl sulfate, sodium polyacrylate, silicate, sodium percarbonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, disodium diaminostilbene disulfonate, amylase, silicone, cellulase, protease variant as described herein;

[0859] - Sodium carbonate, sodium aluminosilicate, sodium sulfate, sodium percarbonate, alkyl sulfate, linear alkylbenzene sulfonate, water, nonanoyloxybenzenesulfonate, sodium polyacrylate, silicate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, Natalase, palmitic acid, amylase, disodium diaminostilbene disulfonate, FD&C Blue 1, silicone, cellulase, alkyl ether sulfate, protease variant as described herein;

[0860] - Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, nonanoyloxybenzenesulfonate, alkyl sulfate, water, silicate, sodium polyacrylate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, amylase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate, protease variant as described herein;

[0861] - Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, alkyl sulfate, water, sodium polyacrylate, silicate, nonanoyloxybenzenesulfonate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, cellulase, amylase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, protease variant as described herein;

[0862] - Water, sodium laureth sulfate, MEA citrate, sodium linear alkylbenzene sulfonate, linear alkylbenzene sulfonate: MEA salt, alcohol ethoxylate, sodium fatty acid, propylene glycol, diethylene glycol, polyethyleneimine ethoxylate propoxylate, diquaternary ammonium ethoxysulfate, ethanol, sodium cumenesulfonate, fragrance, DTPA, sodium bisulfate, disodium diaminostilbene disulfonate, mannanase, cellulase, amylase, sodium formate, calcium formate, laurylamine oxide, Liquitint TM Blue, polydimethylsiloxane / polydimethylsilicone, protease variant as described herein;

[0863] - Water, sodium laureth sulfate, linear alkylbenzene sulfonate: sodium / MEA salt, MEA citrate, propylene glycol, polyethyleneimine ethoxylate, fragrance, ethanol, diethylene glycol, polyethyleneimine propoxyethoxylate, amylase, alcohol sulfate, sodium fatty acid, DTPA, disodium diaminostilbene disulfonate, MEA, mannanase, glucoamylase, sodium formate, polydimethylsiloxane, Liquitint TM Blue, tetraamine, protease variant as described herein;

[0864] - Water, sodium laureth sulfate, MEA citrate, sodium linear alkylbenzene sulfonate, linear alkylbenzene sulfonate: MEA salt, alcohol ethoxylate, sodium fatty acid, propylene glycol, diethylene glycol, polyethyleneimine ethoxylate propoxylate, diquaternary ammonium ethoxysulfate, ethanol, sodium cumenesulfonate, fragrance, DTPA, sodium bisulfate, disodium diaminostilbene disulfonate, mannanase, cellulase, amylase, sodium formate, calcium formate, lauryldimethylamine oxide, Liquitint TM Blue, polydimethylsiloxane / polydimethylsilicone, protease variant as described herein;

[0865] - Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, nonanoyloxybenzenesulfonate, alkyl sulfate, water, silicate, sodium polyacrylate ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, amylase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate, protease variant as described herein; or

[0866] - Water, dodecylbenzenesulfonic acid, laureth-11, peg-75 lanolin, propylene glycol, denatured ethanol (alcoholdenat.), potassium soyate, potassium hydroxide, disodium cocoamphodiacetate, coconut alkylamide edetate, perfume, zinc ricinoleate, sodium chloride, benzisothiazolinone, methylisothiazolinone, ci 16255, benzyl alcohol, protease variant as described herein.

[0867] Preferably, the above formulations comprising protease further comprise 4-FPBA and / or peptide aldehyde protease inhibitors, most preferably Z-GAY or Z-VAL.

[0868] The protease variants described herein can be included in one of the following detergent compositions.

[0869]

[0870]

[0871] %wt in formulation <![CDATA[Linear C 10 C 13 -alkylbenzenesulfonic acid]]> 10.0 <![CDATA[C 12 -C 14 -Fatty alcohol ether sulfate (2EO)]]> 10.0 <![CDATA[C 12 -C 14 -Fatty alcohol ethoxylate (7EO)]]> 20.0 <![CDATA[C 12 -C 18 Coconut fatty acid]]> 5.0 Sodium citrate 1.5 NaOH 50% Propylene glycol 10.0 Formulation comprising the protease variant described herein and optionally one or more enzymes, preferably amylase 0.01％-0.1％ Water Added to 100%

[0872] %wt in formulation <![CDATA[Linear C 10 C 13 -alkylbenzenesulfonic acid]]> 10.0 <![CDATA[C 12 -C 14 -Fatty alcohol ether sulfate (2EO)]]> 10.0 <![CDATA[C 12 -C 14 -Fatty alcohol ethoxylate (7EO)]]> 20.0 <![CDATA[C 12 -C 18 Coconut fatty acid]]> 5.0 MGDA 1.5 NaOH 50% Propylene glycol 10.0 Formulation comprising the protease variant described herein and optionally one or more enzymes, preferably amylase 0.01％-0.1％ Water Added to 100%

[0873] %wt in formulation <![CDATA[Linear C 10 C 13 -alkylbenzenesulfonic acid]]> 10.0 <![CDATA[C 12 -C 14 -Fatty alcohol ether sulfate (2EO)]]> 10.0 <![CDATA[C 12 -C 14 -Fatty alcohol ethoxylate (7EO)]]> 20.0 <![CDATA[C 12 -C 18 Coconut fatty acid]]> 5.0 GLDA 1.5 NaOH 50% Propylene glycol 10.0 Formulation comprising the protease variant described herein and optionally one or more enzymes, preferably amylase 0.01％-0.01％ Water Added to 100%

[0874] %wt in formulation <![CDATA[Linear C 10 C 13 -alkylbenzenesulfonic acid]]> 10.0 <![CDATA[C 12 -C 14 -Fatty alcohol ether sulfate (2EO)]]> 10.0 <![CDATA[C 12 -C 14 -Fatty alcohol ethoxylate (7EO)]]> 20.0 <![CDATA[C 12 -C 18 Coconut fatty acid]]> 5.0 Phosphonate (e.g. HEDP or DTPMP) 1.5 NaOH 50% Propylene glycol 10.0 Formulation comprising the protease variant described herein and optionally one or more enzymes, preferably amylase 0.05％-0.2％ Water Added to 100%

[0875]

[0876]

[0877] Compositions with antimicrobial agents

[0878] 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 that can be added to aqueous products and compositions to maintain the original properties, characteristics and integrity of the products and compositions by killing contaminating microorganisms or inhibiting their growth.

[0879] Compositions / formulations comprising the protease of the present invention may contain one or more antimicrobial agents and / or preservatives as listed on pages 35 to 39 of patent WO 2021 / 115912 A1 (“Formulations comprising a hydrophobically modified polyethyleneimine and one or more enzymes”).

[0880] Of particular interest for cleaning compositions and fabric and home care products and especially in laundry detergent formulations are any of the following antimicrobial agents and / or preservatives:

[0881] 4,4'-dichloro-2-hydroxydiphenyl ether (other names: 5-chloro-2-(4-chlorophenoxy)phenol, hydroxy-dichlorodiphenyl ether (Diclosan), DCPP), HP 100 (30 wt.% DCPP in 1,2 - propanediol); 2 - phenoxyethanol (other names: phenoxyethanol, methyl phenyl glycol, phenoxetol, ethylene glycol phenyl ether, ethylene glycol monophenyl ether, 2 - (phenoxy)ethanol, 2 - phenoxy - 1 - ethanol); 2 - bromo - 2 - nitropropane - 1,3 - diol (other names: 2 - bromo - 2 - nitro - 1,3 - propanediol); glutaraldehyde (other names: 1,5 - pentanedial, pentane - 1,5 - dial, glutaral, glutardialdehyde); glyoxal (other names: ethanedial, oxylaldehyde, 1,2 - ethanedial); 5 - bromo - 5 - nitro - 1,3 - dioxane (other names: 5 - bromo - 5 - nitro - m - dioxane); phenoxypropanol (other names: propylene glycol phenyl ether, phenoxyisopropanol, 1 - phenoxy - 2 - propanol, 2 - phenoxy - 1 - propanol); glucoprotamine (chemical description: reaction product of glutamic acid and alkyl propanediamine, other names: glucoprotamine 50); cyclohexylhydroxydiazene - 1 - oxide, potassium salt (other names: N - cyclohexyl - diazenium dioxide, potassium HDO, Xyligene); formic acid (other names: methanoic acid) and its salts, such as sodium formate; tetrahydro - 3,5 - dimethyl - 1,3,5 - thiadiazine - 2 - thione (other names: 3,5 - dimethyl - 1,3,5 - thiadiazolidine - 2 - thione, dazomet); 2,4 - dichlorobenzyl alcohol (other names: dichlorobenzyl alcohol, 2,4 - dichloro - benzyl alcohol, (2,4 - dichloro - phenyl) - methanol, DCBA); 1 - propanol (other names: n - propanol, propan - 1 - ol, n - propyl alcohol); 1,3,5 - tris(2 - hydroxyethyl) - hexahydro - 1,3,5 - triazine (other names: hexahydrotriazine, tris(hydroxyethyl) - hexahydrotriazine, hexahydro - 1,3,5 - tris(2 - hydroxyethyl) - s - triazine, 2,2′,2″ - (hexahydro - 1,3,5 - triazine - 1,3,5 - triyl)triethanol); 2 - butyl - 1,2 - benzisothiazol - 3(2H) - one (“BBIT”); 2 - methyl - 2H - isothiazol - 3 - one (“MIT”); 2 - octyl - 2H - isothiazol - 3 - one (“OIT”); 5 - chloro - 2 - methyl - 2H - isothiazol - 3 - one (“CIT” or “CMIT”); mixture of 5 - chloro - 2 - methyl - 2H - isothiazol - 3 - one (“CMIT”) and 2 - methyl - 2H - isothiazol - 3 - one (“MIT”) (CMIT / MIT mixture);1,2-Benzisothiazol-3(2H)-one (“BIT”); hex-2,4-dienoic acid (common name “sorbic acid”) and its salts such as calcium sorbate, sodium sorbate; (E,E)-potassium hex-2,4-dienoate (potassium sorbate); lactic acid and its salts; L-(+)-lactic acid; especially sodium lactate; benzoic acid and its salts such as sodium benzoate, ammonium benzoate, calcium benzoate, magnesium benzoate, MEA benzoate, potassium benzoate; salicylic acid and its salts such as calcium salicylate, magnesium salicylate, MEA salicylate, sodium salicylate, potassium salicylate, TEA salicylate; benzalkonium chloride, benzalkonium bromide, benzalkonium saccharinate; didodecyldimethylammonium chloride (“DDAC”); N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (“diamine”); peracetic acid; hydrogen peroxide.

[0882] At least one antimicrobial or preservative can be added to the composition of the present invention at a concentration of 0.001% to 10% relative to the total weight of the composition.

[0883] Preferably, the composition contains 2-phenoxyethanol at a concentration of 0.1% to 2% or 4,4'-dichloro-2-hydroxy diphenyl ether (DCPP) at a concentration of 0.005% to 0.6%.

[0884] The present invention also encompasses a method of preserving an aqueous composition according to the present invention against microbial contamination or growth, the method comprising adding at least one antimicrobial or preservative, preferably 2-phenoxyethanol.

[0885] The present invention also encompasses a method of providing an antimicrobial effect to textiles after treatment with a solid laundry detergent (such as powder, granule, capsule, tablet, stick, etc.), a liquid laundry detergent, a softener or a post-rinse agent containing 4,4'-dichloro-2-hydroxy diphenyl ether (DCPP).

[0886] Usage method

[0887] The protease variants as described herein can be used in various applications.

[0888] The protease variants as described herein or a composition comprising the protease variants as described herein can be used in cleaning, food processing, animal feed, pulp and paper processing, baking, mining and oil well services, textile processing, leather processing, water treatment, brewing, ethanol production, circular economy, waste treatment or recycling.

[0889] The present invention also relates to the use of the protease variants as described herein in a cleaning process (such as laundry washing or hard surface cleaning), preferably for household care or I&I cleaning. The variant polypeptides of the present invention or compositions comprising said variant polypeptides can be used in a variety of industrial and institutional cleaning applications, including commercial laundry washing (such as in-house laundry washing or tunnel washing), mechanical appliance washing (such as in a hood machine or in a tunnel washing machine), manual dishwashing cleaning, carpet cleaning, open plant cleaning (external pipe cleaning), in-place cleaning (internal pipe cleaning), membrane cleaning (such as in dairy, food, beverage or water treatment), vehicle maintenance (such as mat cleaning), microbial removal, virus removal, insect removal, or odor removal or veterinary cleaning. The variant polypeptides of the present invention or compositions comprising said variant polypeptides can also be used in textile processing, leather processing or water treatment.

[0890] The variant polypeptides of the present invention or compositions comprising said variant polypeptides can be used in the bioenergy industry, particularly in bioethanol production, oil and gas recovery (particularly liquefaction for improving oil recovery), and food processing (particularly beverage production and / or processing).

[0891] The present invention also relates to the use of the protease variants as described herein for providing a detergent composition having improved protease stability and / or for providing a detergent composition having improved washing performance (preferably against protease-sensitive stains).

[0892] Accordingly, the present invention also relates to a method for cleaning, preferably laundry washing or hard surface cleaning, which method comprises the step of contacting a test substance, preferably a textile or a hard surface, with a composition comprising a protease variant as described herein, preferably wherein the composition comprises at least one additional detergent component, preferably a surfactant and / or a builder.

[0893] Furthermore, the present invention also relates to a method for improving protease stability in a detergent composition and / or for improving the washing performance of a detergent composition (preferably against protease-sensitive stains), which method comprises the step of formulating a protease variant as described herein in a detergent composition.

[0894] The present invention also relates to a method for laundry washing of fabrics or cleaning hard surfaces, which method comprises treating the fabric or hard surface with a composition comprising a variant polypeptide of the present invention and further comprising 4,4'-dichloro-2-hydroxy diphenyl ether.

[0895] In one embodiment, the protease variants as described herein are used for improving the sustainability characteristics of a composition or method and / or for a circular economy. With regard to use in a circular economy, the protease variants as described herein can be used in waste treatment or recycling.

[0896] In one embodiment, the present invention relates to a method for waste treatment, preferably for dissolving waste, the method comprising the steps of: contacting the waste with a protease variant as described herein under conditions that support the enzymatic dissolution of the waste and preferably with one or more enzymes selected from the group consisting of lipase, dextranase, amylase, pectate lyase, and mannanase. In one embodiment, the waste is municipal solid waste. In one embodiment, the liquefied waste can be used as a substrate for microbial fermentation.

[0897] Although the present invention has been shown and described in detail in the drawings and the foregoing description, such a showing and description should be considered illustrative or exemplary and not restrictive. The present invention is not limited to the disclosed embodiments. By studying the drawings, the present disclosure, and the appended claims, those skilled in the art can understand and implement other variations of the disclosed embodiments when practicing the claimed invention. The detailed description is merely exemplary in nature and is not intended to limit the application and uses. The following examples further illustrate the present invention but do not limit the scope of the present invention. Those skilled in the art can make various changes and modifications based on the description of the present invention, and such changes and modifications are also included in the present invention.

[0898] Preferred embodiment

[0899] Particularly preferred herein is:

[0900] 1. A variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0901] (i) the polypeptide or its fragment has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9% but less than 100% sequence identity, and

[0902] (ii) wherein, as compared to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 and with reference to the numbering of SEQ ID NO:2, the polypeptide contains amino acid substitutions at amino acid residues 43, 78, and 204.

[0903] 2. The variant polypeptide according to Example 1, wherein a fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0904] 3. The variant polypeptide according to Example 1 or 2, wherein:

[0905] (a) 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 X43K;

[0906] (b) the amino acid substitution at amino acid residue 78 is X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, preferably X78N / D; and / or

[0907] (c) the amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably X204D.

[0908] 4. The variant polypeptide according to Examples 1 to 3, with reference to the numbering of SEQ ID NO:2, the variant polypeptide further contains an amino acid substitution at amino acid residue 76, preferably wherein the amino acid substitution at amino acid residue 76 is X76D.

[0909] 5. The variant polypeptide according to any one of the foregoing examples, with reference to the numbering of SEQ ID NO:2, the variant polypeptide further contains an amino acid substitution at amino acid residue 183, preferably wherein the amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, more preferably X183D / E.

[0910] 6. The variant polypeptide according to any one of the foregoing examples, with reference to the numbering of SEQ ID NO:2, the variant polypeptide further contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 109, 144, 182, 237, 240, 248, 256, and 260, preferably wherein:

[0911] (a) the amino acid substitution at amino acid residue 18 is X18A / D / C / E / Q;

[0912] (b) the amino acid substitution at amino acid residue 24 is X24K;

[0913] (c) The amino acid substitution at amino acid residue 56 is X56D;

[0914] (d) The amino acid substitution at amino acid residue 109 is X109K / A;

[0915] (e) The amino acid substitution at amino acid residue 144 is X144N / R;

[0916] (f) The amino acid substitution at amino acid residue 182 is X182K / R / E;

[0917] (g) The amino acid substitution at amino acid residue 237 is X237R / A;

[0918] (h) The amino acid substitution at amino acid residue 240 is X240E / N;

[0919] (i) The amino acid substitution at amino acid residue 248 is X248Q / R;

[0920] (j) The amino acid substitution at amino acid residue 256 is X256E / T / D / R / P; and / or

[0921] (k) The amino acid substitution at amino acid residue 260 is X260D / K.

[0922] 7. The variant polypeptide according to any one of the foregoing embodiments, wherein the polypeptide comprises a combination of substitutions selected from the group consisting of:

[0923] (a) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q

[0924] (b) X24K; X43K; X78N; X109A; X204D

[0925] (c) X24K; X43K; X78N; X204D; X248R; X260K

[0926] (d) X24K; X43K; X78N; X183D; X204D; X248R; X260K

[0927] (e) X24K; X43K; X78N; X204D; X237R; X248R; X260K

[0928] (f) X24K; X43K; X78N; X204D; X240E; X248R; X260K

[0929] (g) X24K; X43K; X78N; X182E; X183D; X204D; X248R; X260K

[0930] (h) X24K; X43K; X78N; X182E; X204D; X237R; X248R; X260K

[0931] (i) X24K; X43K; X78N; X182E; X204D; X240E; X248R; X260K

[0932] (j) X24K; X43K; X78N; X183D; X204D; X237R; X248R; X260K

[0933] (k) X24K; X43K; X78N; X183D; X204D; X240E; X248R; X260K

[0934] (l) X24K; X43K; X78N; X204D; X237R; X240E; X248R; X260K

[0935] (m) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q

[0936] (n) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q

[0937] (o) X24K; X43K; X78N; X156D; X183D; X204D; X240E; X248R; X260K

[0938] (p) X24K; X43K; X78N; X182E; X183D; X204D; X237R; X248R; X260K

[0939] (q) X24K; X43K; X78N; X182E; X183D; X204D; X240E; X248R; X260K

[0940] (r) X24K; X43K; X78N; X182E; X204D; X237R; X240E; X248R; X260K

[0941] (s) X24K; X43K; X78N; X183D; X204D; X237R; X240E; X248R; X260K

[0942] (t) X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q

[0943] (u) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260K

[0944] (v) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X248Q

[0945] (w) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260D

[0946] (x) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260K

[0947] (y) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X240E; X248Q

[0948] (z) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260D

[0949] (aa) X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q; X260D

[0950] (bb) X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D

[0951] (cc) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q

[0952] (dd) X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D

[0953] (ee) X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X240E; X248Q; X260K

[0954] (ff) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q; X260D

[0955] (gg) X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R

[0956] (hh) X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R

[0957] (ii) X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R, and

[0958] (jj) X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X237A; X240E; X248R.

[0959] 8. A variant polypeptide according to any one of the foregoing embodiments, wherein the polypeptide comprises a combination of substitutions selected from the group consisting of:

[0960] (a) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0961] (b) S24K; N43K; S78N; Q109A; N204D

[0962] (c) S24K; N43K; S78N; N204D; N248R; T260K

[0963] (d) S24K; N43K; S78N; N183D; N204D; N248R; T260K

[0964] (e) S24K; N43K; S78N; N204D; K237R; N248R; T260K

[0965] (f) S24K; N43K; S78N; N204D; S240E; N248R; T260K

[0966] (g) S24K; N43K; S78N; Q182E; N183D; N204D; N248R; T260K

[0967] (h) S24K; N43K; S78N; Q182E; N204D; K237R; N248R; T260K

[0968] (i) S24K; N43K; S78N; Q182E; N204D; S240E; N248R; T260K

[0969] (j) S24K; N43K; S78N; N183D; N204D; K237R; N248R; T260K

[0970] (k) S24K; N43K; S78N; N183D; N204D; S240E; N248R; T260K

[0971] (l) S24K; N43K; S78N; N204D; K237R; S240E; N248R; T260K

[0972] (m) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q

[0973] (n) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q

[0974] (o) S24K; N43K; S78N; S156D; N183D; N204D; S240E; N248R; T260K

[0975] (p) S24K; N43K; S78N; Q182E; N183D; N204D; K237R; N248R; T260K

[0976] (q) S24K; N43K; S78N; Q182E; N183D; N204D; S240E; N248R; T260K

[0977] (r) S24K; N43K; S78N; Q182E; N204D; K237R; S240E; N248R; T260K

[0978] (s) S24K; N43K; S78N; N183D; N204D; K237R; S240E; N248R; T260K

[0979] (t) S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q

[0980] (u) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260K

[0981] (v) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; N248Q

[0982] (w) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260D

[0983] (x) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260K

[0984] (y) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; S240E; N248Q

[0985] (z) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260D

[0986] (aa) S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q; T260D

[0987] (bb) S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D

[0988] (cc) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q

[0989] (dd) S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D

[0990] (ee) S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; S240E; N248Q; T260K

[0991] (ff) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D

[0992] (gg) V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R

[0993] (hh) V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R

[0994] (ii) V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R, and

[0995] (jj) V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R.

[0996] 9. A variant polypeptide according to any one of the foregoing embodiments, wherein, with reference to the numbering of SEQ ID NO: 2, the polypeptide comprises the amino acid residue D or E at position 101, preferably E at position 101.

[0997] 10. A variant polypeptide according to any one of the foregoing embodiments, wherein the polypeptide does not comprise the amino acid substitutions S3T, V4I and V199I.

[0998] 11. A variant polypeptide according to any one of the foregoing embodiments, wherein, compared to the protease shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, the polypeptide exhibits one or more improved properties, preferably wherein these improved properties are selected from:

[0999] (i) increased stability,

[1000] (ii) increased storage stability, and

[1001] (iii) Increased storage stability in detergent compositions.

[1002] 12. A polynucleotide that encodes a variant polypeptide as described in any one of the foregoing examples.

[1003] 13. A formulation that comprises a variant polypeptide as described in any one of Examples 1 to 11 and at least one additional component.

[1004] 14. The formulation as described in Example 13, wherein the formulation comprises an enzyme stabilization system, and wherein the enzyme stabilization system preferably comprises at least one compound selected from the group consisting of: polyols (preferably, 1,3-propanediol, ethylene glycol, glycerol, 1,2-propanediol, or sorbitol), inorganic salts (preferably, CaCl2, MgCl2, or NaCl), short-chain (preferably, C1-C3) carboxylic acids or their salts (preferably, formic acid, formates (preferably, sodium formate), acetic acid, acetates, or lactates), borates, boric acid, boronic acid (preferably, 4-formylphenylboronic acid (4-FPBA)), peptide aldehydes (preferably, Z-VAL-H or Z-GAY-H), peptide acetals, and peptide aldehyde bisulfite adducts, preferably peptide aldehydes (preferably, Z-VAL-H or Z-GAY-H).

[1005] 15. The formulation as described in Example 13 or 14, wherein the formulation comprises one or more second enzymes different from the variant polypeptides mentioned in any one of the foregoing examples, preferably one or more second enzymes selected from the group consisting of: amylases, second proteases, lipases, cellulases, hemicellulases, mannanases, xylanases, DNases, dispersins, pectinases, redox enzymes, and cutinases, preferably selected from amylases, mannanases, and lipases, most preferably amylase.

[1006] 16. A detergent composition that comprises a variant polypeptide as described in any one of Examples 1 to 11, preferably a laundry detergent composition or a hard surface cleaning detergent composition.

[1007] 17. The detergent composition as described in Example 16, wherein the composition comprises one or more surfactants and / or one or more builders, preferably strong chelating builders.

[1008] 18. The detergent composition as described in Example 17, wherein the composition comprises a builder, and wherein the builder is selected from MDGA, GLDA, DTPMP, HEDP, and EDDS, preferably MDGA or EDDS.

[1009] 19. The detergent composition according to embodiment 17, wherein the composition comprises a surfactant, and the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and combinations thereof.

[1010] 20. The detergent composition according to any one of embodiments 16 to 19, wherein the detergent composition does not contain anionic surfactants.

[1011] 21. The detergent composition according to any one of embodiments 17 to 20, wherein the surfactant and / or the builder is biodegradable and / or bio-based.

[1012] 22. The detergent composition according to any one of embodiments 16 to 21, wherein the detergent composition is liquid or solid.

[1013] 23. The detergent composition according to any one of embodiments 16 to 22, wherein the detergent composition is in the form of a sachet.

[1014] 24. The detergent composition according to any one of embodiments 16 to 23, wherein the detergent composition is a liquid laundry detergent composition.

[1015] 25. The detergent composition according to any one of embodiments 16 to 24, wherein the detergent composition does not contain boron.

[1016] 26. The detergent composition according to any one of embodiments 16 to 25, wherein the detergent composition does not contain a preservative.

[1017] 27. The detergent composition according to any one of embodiments 16 to 25, wherein the composition further comprises 2-phenoxyethanol, preferably comprising phenoxyethanol in an amount ranging from 2 ppm to 5% by weight of the composition; more preferably comprising 0.1% to 2% of phenoxyethanol.

[1018] 28. The detergent composition according to any one of embodiments 16 to 27, wherein the composition further comprises 4,4'-dichloro-2-hydroxy diphenyl ether at a concentration of 0.001% to 3%, preferably 0.002% to 1%, more preferably 0.01% to 0.6%, each by weight of the composition.

[1019] Examples

[1020] Materials and methods

[1021] 1. Library generation

[1022] The gene encoding the protease was cloned into a Gram-positive expression vector by a standard protocol based on restriction endonuclease digestion-ligation. The vector contains a promoter sequence, a sequence encoding a secretion signal peptide, and a ribosome binding site. After the reaction, the plasmid assembly mixture was transformed into Bacillus subtilis PY79 by an established natural competence transformation method. Successful transformation was selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubating overnight at 37 °C. After overnight selection, individual colonies were grown overnight by shaking at 1000 rpm at 35 °C in TB medium with 20 μg / ml kanamycin sulfate. The cells were then pelleted by centrifugation, and plasmid DNA was isolated by alkaline lysis using the QIAprep Spin Miniprep Kit from Qiagen. The isolated DNA was transformed into electrocompetent Bacillus licheniformis cells. For example, the cells were prepared by growing the Bacillus licheniformis strain in a medium rich in concentrated osmolyte (such as LB broth with 0.5 M D-sorbitol) and harvesting the cells in the early exponential growth phase. The cells were harvested by cooling on ice and pelleting by centrifugation. After harvesting, the cells were washed by three suspension-precipitation (by centrifugation) cycles with a washing buffer rich in osmolyte (such as 10% glycerol with 0.5 M D-sorbitol and 0.5 M D-mannitol) to remove salts. Finally, the cells were concentrated by resuspending in the washing buffer at 1% to 10% of the original culture volume. Once prepared, the plasmid DNA was added to the electrocompetent Bacillus licheniformis cells in a 0.2 cm electroporation cuvette from Bio-Rad. The cells were electroporated using the Gene Pulser Xcell from Bio-Rad according to the manufacturer's instructions. The cells were immediately rescued by adding 1 ml of medium rich in concentrated osmolyte after the pulse. After recovery at 37 °C for two hours, successful transformants were selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubating overnight at 37 °C.

[1023] 2. Preparation and expression of variants

[1024] Individual colonies of the expression strain were picked into 600 μL of rich medium (such as LB broth) supplemented with 20 μg / mL kanamycin sulfate in a 96-well plate. The cultures were grown by shaking at 1000 rpm at 30 °C for 16 hours, and then 6 μL of the culture was used to inoculate 600 μL of defined glucose-mineral medium and 20 μg / mL kanamycin sulfate in the 96-well plate. The cultures were grown by shaking at 1000 rpm at 30 °C for 48 hours, and then the supernatant was harvested by pelleting the cells by centrifugation and removing the remaining culture broth.

[1025] 3. Protease activity assay

[1026] Proteolytic activity was determined by using the Suc-AAPF-AMC substrate (Bachem, product number: 4012873). Suc-AAPF-AMC is the abbreviation of N-succinyl-alanine-alanine-proline-phenylalanine-7-amino-4-methylcoumarin, which is a blocked peptide that can be cleaved by endoproteases. After proteolytic cleavage, free AMC molecules are released and measured by fluorospectrophotometry at an excitation wavelength of 360 nm and an emission wavelength of 448 nm. Under a given set of conditions, the slope (Vmax) of the time-dependent increase in the fluorescence signal is proportional to the amount of protease in the solution and the specific activity of the protease under discussion (activity per mg of enzyme). Before activity assessment, protease samples were diluted in assay buffer (100 mM Tris-HCl, 0.1% Brij-35, pH 8.6) or detergent excitation solution, for example, to a pre-assay dilution factor between 400 - 800-fold. The assay was performed by transferring 10 μL of the diluted enzyme sample to a 384-well microtiter plate containing 40 μL of the substrate working solution or by transferring 20 μL of the diluted enzyme sample to a 96-well microtiter plate containing 80 μL of the substrate working solution. The solutions were mixed at room temperature and the fluorescence signal was measured every minute for 15 minutes at an excitation wavelength of 360 nm and an emission wavelength of 448 nm using a standard fluorescence-capable microplate reader (Synergy from BioTek) or ). The substrate conversion rate (V max ) of each sample was calculated, and the residual activity was calculated by dividing the activity after storage time by the activity of the sample at time point zero. In some cases, the residual activity was normalized relative to the residual activity of a specific reference sample and expressed as the normalized residual activity.

[1027] Example 1: Conditions for performing accelerated stability studies in standard detergents

[1028] To evaluate the stability in detergents, the expressed protease supernatant was first diluted 10 - 15 fold into a stability buffer (20 mM HEPES, pH 8.0) containing a limited amount of calcium (Table 3), and added to a standard liquid detergent (Standard A or B, Table 4), equilibrated for 1 - 2 hours, and then the activity at time zero was evaluated. Then it was stored in the liquid detergent (Standard A or B) at 37 or 45 degrees Celsius, and the residual activity was evaluated after 1, 2, 3, and / or 7 days. Before activity evaluation by Suc - AAPF - AMC assay as described in the Materials and Methods section, the protease samples excited in detergents were pre - diluted 5 - 40 fold in an activity buffer (100 mM Tris - HCl, 0.1% Brij - 35, pH 8.6).

[1029] Table 3. Excitation conditions for accelerated stability studies

[1030]

[1031]

[1032] Table 4. Standard detergents for accelerated stability studies

[1033]

[1034] Example 2: Evaluation of accelerated storage stability of mutants from a combinatorial library under Condition 1

[1035] Mutants from SEQ ID NO:3 were generated by a combinatorial method, and a portion of the library was expressed in 96 - well plates as described in the Materials and Methods. The supernatant containing the protease was excited under Condition 1 (see Example 1), subjected to thorough mixing, and equilibrated for 1 h. The activity of the non - stressed sample was measured by Suc - AAPF - AMC assay. The detergent plates were sealed and placed at an elevated temperature (37 degrees Celsius) for a duration of 18 - 160 hours. At designated time points during the incubation, the protease activity of the stressed samples was measured and compared to the non - stressed control to calculate the residual activity. SEQ ID NO:3 was included as a reference control.

[1036] Compared to SEQ ID NO:3, the combinatorial library based on SEQ ID NO:3 generated variants containing 2 - 19 mutations at different selected positions. Progressive and cooperative stabilization of the variants was observed, with an increase in the number of mutations from a selected core set of stabilizing mutations consisting of N43K, S78N / D, and N204D (Table 5).

[1037] Table 5

[1038]

[1039]

[1040] *The residual activity was defined as the activity after storage at 37 °C for 160 h divided by the activity at time point zero.

[1041] Example 3: Accelerated storage stability determination of mutants from a single-site mutagenesis library under Condition 2

[1042] A single-site mutagenesis library was constructed from SEQ ID NO:39, which contained three stabilizing core group positions (N43K, S78N, and N204D). Each position in the core group was reverted to SEQ ID NO:3. The mutants were expressed according to the protocol listed in Materials and Methods, and the supernatant containing the protease was excited under Condition 2 (see Example 1), subjected to thorough mixing, and equilibrated for 2 h. The activity of the non-stressed sample was measured using a Suc-AAPF-AMC assay. The detergent plates were sealed and placed at an elevated temperature (45 °C) for a duration of 44 h. The protease activity of the stressed sample was measured and compared to the non-stressed control to calculate the residual activity. The protease according to SEQ ID NO:39 was included as a reference control. Stabilization (increased residual activity compared to SEQ ID NO:3) was significantly reduced in variants with reverse mutations that had residue identity to the residues present in SEQ ID NO:3 (Table 6), which supported the role of the stabilizing core group.

[1043] Table 6

[1044]

[1045] *Residue identity to SEQ ID NO:3 at the indicated position

[1046] **Normalized value of the residual activity relative to SEQ ID NO:39

[1047] Example 4: Accelerated storage stability assessment of mutants from a combinatorial library under Condition 3

[1048] Mutants from SEQ ID NO:3 were generated by combinatorial methods and a portion of the library was expressed in 96DWP as described in Materials and Methods. The supernatant containing the protease was excited under Condition 3 (see Example 1), subjected to thorough mixing, and equilibrated for 1 h. The activity of the non-stressed sample was measured using the Suc-AAPF-AMC assay. The detergent plates were sealed and placed at an elevated temperature (45 degrees Celsius) for a duration of 18 - 160 hours. At specified time points during the incubation, the protease activity of the stressed samples was measured and compared to the non-stressed control to calculate the residual activity. SEQ ID NO:3 was included as a reference control. The residual activities of the selected variants and the reference control are reported in Table 7.

[1049] Table 7

[1050]

[1051]

[1052] *Residual activity is defined as the activity after storage for 160 h under Condition 3 divided by the activity at time point zero

[1053] Example 5: Stable Detergent Formulations

[1054] Liquid laundry detergent formulations were prepared with or without (control) 0.5% by weight of the variant polypeptide of the present invention and 0.2% biocide HP 100 (from BASF SE) or 1% 2-phenoxyethanol ( PE, BASF SE). These formulations were prepared by first preparing a premix containing AEO and AES surfactants, the solvents 1,2-propanediol and ethanol, and (where relevant) HP 100 or 2-phenoxyethanol. The premix was stirred at room temperature to form a homogeneous mixture. Then, LAS, fatty acids, and citric acid as shown in Table 8 and water were added up to 90%. Subsequently, the pH was adjusted to pH = 8.5 using NaOH. The final formulation was then prepared by stirring at room temperature: 90% of this obtained mixture, 0.5% of the polypeptide of the present invention, and water up to 100%.

[1055] The compositions and results are shown in Table 8.

[1056] Table 8

[1057]

[1058] AEO: C 13 / C 15 oxo-alcohol (7EO) AO7 (BASF SE) (CAS 68002-97-1)

[1059] AES: C 12 / C 14 -Fatty alcohol ether sulfate (2EO), sodium salt: N 70 (BASF SE) (CAS 68891-38-3)

[1060] LAS: Linear alkylbenzene sulfonic acid DBS / LC (BASF SE) (CAS 85536-14-7)

[1061] Coconut fatty acid: K12-18 (Emery Oleochemicals) (CAS 90990-15-1)

[1062] 1,2-Propanediol: racemic mixture (CAS 57-55-6)

[1063] HP 100 is a commercial product from BASF SE and contains 30% antimicrobial active 4,4'-dichloro-2-hydroxy diphenyl ether (CAS 3380-30-1) in 1,2-propanediol (CAS 122-99-6).

[1064] 2-Phenoxyethanol can be used as PE obtained from BASF SE

[1065] The concentrations of surfactant products are given in the table above.

[1066] It can be clearly seen from the table above that the polypeptide of the present invention can be combined with Tinosan HP 100 or 2-phenoxyethanol in a liquid laundry detergent formulation.

Claims

1. A variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein: (i) the polypeptide or its fragment has an amino acid sequence having at least 60% but less than 100% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and (ii) compared with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 and with reference to the numbering of SEQ ID NO:2, the polypeptide contains amino acid substitutions at amino acid residues 43, 78 and 204.

2. The variant polypeptide according to claim 1, wherein: (a) 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 X43K; (b) the amino acid substitution at amino acid residue 78 is X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, preferably X78N / D; and / or (c) the amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably X204D.

3. The variant polypeptide according to claim 1 or 2, with reference to the numbering of SEQ ID NO:2, the variant polypeptide further contains an amino acid substitution at amino acid residue 76, preferably wherein the amino acid substitution at amino acid residue 76 is X76D.

4. The variant polypeptide according to any one of the preceding claims, with reference to the numbering of SEQ ID NO:2, the variant polypeptide further contains an amino acid substitution at amino acid residue 183, preferably wherein the amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, more preferably X183D / E.

5. The variant polypeptide according to any one of the preceding claims, with reference to the numbering of SEQ ID NO:2, the variant polypeptide further contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 109, 144, 182, 237, 240, 248, 256 and 260, preferably wherein ： (a) the amino acid substitution at amino acid residue 18 is X18A / D / C / E / Q; (b) the amino acid substitution at amino acid residue 24 is X24K; (c) the amino acid substitution at amino acid residue 56 is X56D; (d) the amino acid substitution at amino acid residue 109 is X109K / A; (e) the amino acid substitution at amino acid residue 144 is X144N / R; (f) the amino acid substitution at amino acid residue 182 is X182K / R / E; (g) the amino acid substitution at amino acid residue 237 is X237R / A; (h) the amino acid substitution at amino acid residue 240 is X240E / N; (i) the amino acid substitution at amino acid residue 248 is X248Q / R; (j) the amino acid substitution at amino acid residue 256 is X256E / T / D / R / P; and / or (k) The amino acid substitution at amino acid residue 260 is X260D / K.

6. A variant polypeptide according to any one of the preceding claims, wherein the polypeptide comprises a combination of substitutions selected from the group consisting of: (a) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q (b) X24K; X43K; X78N; X109A; X204D (c) X24K; X43K; X78N; X204D; X248R; X260K (d) X24K; X43K; X78N; X183D; X204D; X248R; X260K (e) X24K; X43K; X78N; X204D; X237R; X248R; X260K (f) X24K; X43K; X78N; X204D; X240E; X248R; X260K (g) X24K; X43K; X78N; X182E; X183D; X204D; X248R; X260K (h) X24K; X43K; X78N; X182E; X204D; X237R; X248R; X260K (i) X24K; X43K; X78N; X182E; X204D; X240E; X248R; X260K (j) X24K; X43K; X78N; X183D; X204D; X237R; X248R; X260K (k) X24K; X43K; X78N; X183D; X204D; X240E; X248R; X260K (l) X24K; X43K; X78N; X204D; X237R; X240E; X248R; X260K (m) X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q (n) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q (o) X24K; X43K; X78N; X156D; X183D; X204D; X240E; X248R; X260K (p) X24K; X43K; X78N; X182E; X183D; X204D; X237R; X248R; X260K (q) X24K; X43K; X78N; X182E; X183D; X204D; X240E; X248R; X260K (r) X24K; X43K; X78N; X182E; X204D; X237R; X240E; X248R; X260K (s) X24K; X43K; X78N; X183D; X204D; X237R; X240E; X248R; X260K (t)X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q (u)X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260K (v)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X248Q (w)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260D (x)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X248Q; X260K (y)X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X240E; X248Q (z)X24K; X43K; X56D; X78N; X144N; X182E; X183D; X204D; X248Q; X260D (aa)X24K; X43K; X56D; X78N; X109K; X144N; X182E; X183D; X204D; X248Q; X260D(bb)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D(cc)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q(dd)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D(ee)X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X240E; X248Q; X260K(ff)X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q; X260D(gg)X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R(hh)X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R(ii)X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R, and (jj)X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X237A; X240E; X248R.

7. A variant polypeptide as described in any one of the preceding claims, wherein the polypeptide comprises a combination of substitutions selected from the group consisting of: (a) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q (b) S24K; N43K; S78N; Q109A; N204D (c) S24K; N43K; S78N; N204D; N248R; T260K (d) S24K; N43K; S78N; N183D; N204D; N248R; T260K (e) S24K; N43K; S78N; N204D; K237R; N248R; T260K (f) S24K; N43K; S78N; N204D; S240E; N248R; T260K (g) S24K; N43K; S78N; Q182E; N183D; N204D; N248R; T260K (h) S24K; N43K; S78N; Q182E; N204D; K237R; N248R; T260K (i) S24K; N43K; S78N; Q182E; N204D; S240E; N248R; T260K (j) S24K; N43K; S78N; N183D; N204D; K237R; N248R; T260K (k) S24K; N43K; S78N; N183D; N204D; S240E; N248R; T260K (l) S24K; N43K; S78N; N204D; K237R; S240E; N248R; T260K (m) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q (n) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q (o) S24K; N43K; S78N; S156D; N183D; N204D; S240E; N248R; T260K (p) S24K; N43K; S78N; Q182E; N183D; N204D; K237R; N248R; T260K (q) S24K; N43K; S78N; Q182E; N183D; N204D; S240E; N248R; T260K (r) S24K; N43K; S78N; Q182E; N204D; K237R; S240E; N248R; T260K (s) S24K; N43K; S78N; N183D; N204D; K237R; S240E; N248R; T260K (t) S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q (u) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260K (v) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; N248Q (w) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260D (x) S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; N248Q; T260K (y) S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; S240E; N248Q (z)S24K; N43K; S56D; S78N; S44N; Q182E; N183D; N204D; N248Q; T260D (aa)S24K; N43K; S56D; S78N; Q109K; S44N; Q182E; N183D; N204D; N248Q; T260D (bb)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D (cc)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q; (dd)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; N248Q; T260D (ee)S24K; N43K; S56D; S78N; Q109K; S44N; Q182K; N183D; N204D; S240E; N248Q; T260K(ff)S24K; N43K; S56D; S78N; S44N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D(gg)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R(hh)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R(ii)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R, and (jj)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R。 8. A variant polypeptide as claimed in any one of the preceding claims, wherein, with reference to the numbering of SEQ ID NO:2, the polypeptide comprises an amino acid residue D or E at position 101, preferably E at position 101.

9. A variant polypeptide as claimed in any one of the preceding claims, wherein the polypeptide exhibits one or more improved properties as compared to the protease shown in SEQ ID NO:1 or SEQ ID NO:3, preferably SEQ ID NO:1, preferably wherein said improved properties are selected from: (i) increased stability, (ii) increased storage stability, and (iii) increased storage stability in a detergent composition.

10. A polynucleotide that encodes a variant polypeptide as described in any one of the preceding claims.

11. A composition comprising the variant polypeptide as described in any one of claims 1 to 9 and at least one additional component, preferably wherein the composition comprises an enzyme stabilization system, wherein the enzyme stabilization system preferably comprises at least one compound selected from the group consisting of: polyols (preferably, 1,3-propanediol, ethylene glycol, glycerol, 1,2-propanediol or sorbitol), inorganic salts (preferably, CaCl 2 , MgCl 2 or NaCl), short-chain (preferably, C1-C3) carboxylic acids or their salts (preferably, formic acid, formates (preferably, sodium formate), acetic acid, acetates, or lactates), borates, boric acid, boronic acid (preferably, 4-formylphenylboronic acid (4-FPBA)), peptide aldehydes (preferably, Z-VAL-H or Z-GAY-H), peptide acetals and peptide aldehyde bisulfite adducts, preferably peptide aldehydes (preferably, Z-VAL-H or Z-GAY-H).

12. The composition according to claim 11, wherein the composition comprises one or more second enzymes different from the variant polypeptide mentioned in any one of the preceding claims, preferably one or more second enzymes selected from the group consisting of: amylase, a second protease, lipase, cellulase, hemicellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase, and cutinase, preferably selected from amylase, mannanase, and lipase, most preferably amylase.

13. The composition according to claim 11 or 12, wherein the composition is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition, preferably wherein the composition comprises one or more surfactants and / or one or more builders, preferably a strong chelating builder.

14. The composition according to any one of claims 11 to 13, wherein the composition further comprises 2-phenoxyethanol and / or 4,4'-dichlor-2-hydroxy-diphenyl ether, preferably comprising phenoxyethanol in an amount ranging from 2 ppm to 5% by weight of the composition; more preferably comprising 0.1% to 2% by weight of phenoxyethanol and / or preferably comprising 4,4'-dichlor-2-hydroxy-diphenyl ether at a concentration of 0.001% to 3%, preferably 0.002% to 1%, more preferably 0.01% to 0.6%, each by weight of the composition.

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