Protease-containing detergent composition with increased storage stability

Abstract

The invention relates to a detergent composition comprising a protease, characterized in that the amino acid sequence of the protease exhibits 60% or more identity with one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. The detergent composition exhibits high storage stability with respect to the activity of the protease. The invention further relates to a method for producing a detergent composition with improved storage stability by adding a protease to the detergent composition, characterized in that the amino acid sequence of the protease exhibits 60% or more identity with one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. The invention also relates to a method for cleaning items, in particular for cleaning dishes or textiles, by bringing the items into contact with the detergent composition according to the invention.

Description

field of technology

[0001] The invention relates to protease-containing detergent compositions, in particular protease-containing dishwashing detergents and textile detergents. Current state of the art

[0002] Proteases are an important component of modern detergent formulations. They are used particularly in dishwashing detergents and fabric detergents. Proteases catalyze the hydrolytic cleavage of peptide bonds and can thus effectively dissolve protein- and peptide-containing soils.

[0003] Of particular economic importance are serine proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62). They hydrolyze peptide bonds within polypeptides with high efficiency and have a very broad spectrum of potential substrates. A large number of different subtilisins and similar enzymes have been isolated from bacteria of the genus Bacillus, particularly the species B. subtilis (subtilisin E), B. lentus, B. licheniformis (Subtilisin Carlsberg, Alcalase®), and B. amylo-lique-faciens, but also from the molds Tritirachium album and Thermoactinomyces vulgaris. An example of a well-known subtilisin-type protease is savinase, derived from Bacillus lentus.

[0004] One problem with the use of proteases in detergent compositions is the limited storage stability of the protease. Experience has shown that the protease loses some of its activity when stored for extended periods of several weeks at temperatures of 30°C or more. This loss of activity may be caused by mutual proteolysis of the protease molecules in the composition, interaction of the protease with any complexing agents in the detergent composition, and / or denaturation of the protease as a result of interaction with other components of the detergent composition.

[0005] There is therefore a need for proteases that are stable in detergent compositions over a longer period of time. Technical task

[0006] The object of the invention is to provide a protease-containing detergent composition with increased storage stability. Storage stability is measured based on the protease activity. The protease activity in the detergent composition should remain stable even when stored for a period of two or more weeks at a temperature of 40°C. Description of the invention

[0007] Surprisingly, it was found that a protease derived from Alkalibacillus haloalkaliphilus having an amino acid sequence according to SEQ ID NO: 1, a protease derived from Pontibacillus marinus having the amino acid sequence according to SEQ ID NO: 2, a protease derived from Litchfieldia alkalitelluris having the amino acid sequence according to SEQ ID NO: 3, or a variant of these proteases sufficiently similar with respect to one of these amino acid sequences exhibits excellent stability in detergent compositions. This finding was particularly surprising because the proteases according to the invention were previously unknown for use in detergents.

[0008] One aspect of the invention therefore relates to a detergent composition comprising a protease, characterized in that the amino acid sequence of the protease has an identity of 60% or more to one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0009] Preferably, the activity of the protease in the detergent composition after storage of the detergent composition at a temperature of 40°C for a period of two weeks is at least 90% of the activity of the protease immediately after preparation of the detergent composition.

[0010] Preferably, the detergent composition contains 0.0001 wt% to 5 wt% of the protease, based on the total mass of active protease and the total mass of the detergent composition.

[0011] Preferably, the detergent composition has a pH in the range of 7.5 to 9.

[0012] Preferably, the detergent composition comprises 10 wt% to 70 wt% anionic and / or non-ionic surfactants, 5 wt% to 50 wt% polyols, 0.1 wt% to 5 wt% complexing agent, and up to 50% water by weight.

[0013] Preferably, the detergent composition comprises at least one polyol having 3 to 6 carbon atoms and 2 to 3 OH groups.

[0014] Preferably, the detergent composition comprises at least one alkylbenzenesulfonic acid and / or at least one alkoxylated fatty alcohol.

[0015] Preferably, the detergent composition comprises no more than 1% by weight of boric acid and borates, based on the total mass of the detergent composition.

[0016] One aspect of the invention relates to a process for producing a detergent composition with improved storage stability by adding a protease to the detergent composition, characterized in that the amino acid sequence of the protease has an identity of 60% or more to the amino acid sequence according to SEQ ID NO: 1.

[0017] One aspect of the invention relates to a method for cleaning laundry, in particular for cleaning dishes or textiles, by bringing the laundry into contact with the detergent composition according to the invention. Advantageous effects of the invention

[0018] The detergent composition according to the invention exhibits excellent storage stability with respect to protease activity. The protease activity in the detergent composition is at least 90% of the activity immediately after preparation of the detergent composition, even when stored for at least two weeks at a temperature of 30°C or 40°C. The protease according to the invention thus proves to be more stable than comparable proteases, such as Savinase. Description of the embodiments

[0019] Embodiments of the invention will be described in detail below. However, the invention is not limited to the following embodiments.

[0020] One aspect of the invention relates to a detergent composition. The detergent composition is used for cleaning laundry. For the purposes of the invention, laundry includes, for example, hard surfaces such as stone floors, metal surfaces, glass surfaces or wooden surfaces, dishes or products made of fibrous material. In the broadest sense, the laundry can also be body surfaces such as hands. The detergent composition can be, for example, a floor cleaner, a toilet cleaner, a dishwashing detergent, a textile detergent or a handwashing detergent. In one embodiment, the detergent composition is used as a dishwashing detergent for cleaning all types of dishes and cutlery made of stainless steel, porcelain, glass or plastic. In an alternative embodiment, the detergent composition is used for cleaning all types of woven, knitted or warp-knitted textiles, as well as furs.In particular, the detergent composition can be used to clean laundry made of natural and / or synthetic fibers, for example, cotton, wool, linen, polyamide, polyester, polyacrylonitrile, or blends thereof. For example, the detergent composition serves as a textile detergent for cleaning laundry, such as clothing, towels, bed linen, blankets, and curtains.

[0021] Cleaning refers to the removal of soiling from the laundry. This soiling can include, in particular, solid pigmented soiling, such as dust, soot, and metal abrasion; greasy and oily soiling, such as skin grease, cooking fat, and lubricating oil; colored soiling, such as fruit or plant juice and blood; and water-soluble soiling, such as urea, sugar, and salt.

[0022] The detergent composition is particularly suitable for being diluted with water to form a wash bath which can be brought into contact with the laundry to be cleaned in order to loosen and wash away soiling.

[0023] The detergent composition can be used for both manual and machine washing. The detergent composition can be used, in particular, as a general-purpose detergent for all washing processes and temperatures. It can also be used as a washing aid together with another general-purpose detergent to extend and / or enhance its effect. It can also be used before or after treatment with another detergent, for example, to loosen soiling before a main wash cycle or to impart further desirable properties to the laundry after a main wash cycle, such as shine, pleasant smell, pleasant feel, crease resistance, or low static charge.

[0024] The detergent composition comprises, as an essential ingredient, a protease having an amino acid sequence that has an identity of 60% or more to one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. The protease has been shown to have excellent storage stability in the detergent composition described herein.

[0025] The protease is particularly preferably the protease according to SEQ ID NO: 1 or a variant thereof with an identity of 60% or more to SEQ ID NO: 1.

[0026] Preferably, the amino acid sequence of the protease has an identity of 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The variants of SEQ ID NO: 1 are particularly suitable for use in detergent compositions.

[0027] In a preferred embodiment, the amino acid sequence of the protease has an identity of 70% or more, preferably 80% or more, further preferably 85% or more, particularly preferably 90% or more, most preferably 95% or more to one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The variants of SEQ ID NO: 1 are particularly suitable for use in detergent compositions.

[0028] In a particularly preferred embodiment, the amino acid sequence of the protease has an identity of 70% or more, preferably 80% or more, more preferably 85% or more, particularly preferably 90% or more, most preferably 95% or more to the amino acid sequence according to SEQ ID NO: 1.

[0029] In a particularly preferred embodiment, the amino acid sequence of the protease is identical to one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. The protease according to SEQ ID NO: 1 is particularly suitable for use in detergent compositions

[0030] Within the scope of the invention, the identity between two amino acid sequences is determined by pairwise alignment using the Needleman-Wunsch algorithm. An exemplary implementation of this algorithm is provided by the European Bioinformatics Institute (EMBL-EBI) (Madeira F, Pearce M, Tivey ARN, et al. "Search and sequence analysis tools services from EMBL-EBI in 2022", Nucleic Acids Research, 2022). The alignment is calculated using the BLOSUM62 substitution matrix, a gap open cost of 10, and a gap extension cost of 0.5. As a measure of identity, the percentage ratio of identical amino acids in the alignment to the total number of opposing amino acids in the alignment is calculated according to the following formula: (number of identical amino acids in the alignment) / (total length of the alignment - number of gaps in the alignment).

[0031] The protease can be produced, in particular, by recombinant protein expression in Bacillus subtilis DB104 using a pFF-RED expression plasmid. A suitable production method is described in the prior art (Falkenberg F et al., "New robust subtilisins from halotolerant and halophilic Bacillaceae," Appl Microbiol Biotechnol, 2023; Falkenberg F et al., "Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T," FEBS Open Bio, 2022).

[0032] The protease according to the invention is characterized in that its activity in a detergent composition remains stable even when stored at an elevated temperature of 40°C over a period of several weeks. Compared to other proteases, the protease exhibits improved stability in the presence of other components of the detergent composition.

[0033] The detergent composition is characterized in particular in that the activity of the protease in the detergent composition is retained for a period of at least two weeks, preferably at least four weeks, after storage of the detergent composition at a temperature of 40 °C.

[0034] Preferably, the activity of the protease in the detergent composition after storage of the detergent composition at a temperature of 40°C for a period of two weeks is at least 90%, preferably at least 95%, more preferably at least 98%, particularly preferably at least 99% of the activity of the protease immediately after preparation of the detergent composition. This activity is preferably also achieved after storage for a period of four weeks. For this purpose, the activity immediately after preparation of the detergent composition should be determined no later than 12 hours after preparation of the detergent composition, preferably no later than 6 hours, particularly preferably no later than 2 hours after preparation.

[0035] In this context, the protease activity is determined by hydrolysis of a test substrate. The preferred substrate for this purpose is N-succinyl-alanine-alanine-proline-phenylalanine-para-nitroanilide, whose cleavage can be measured by the released para-nitroaniline chromophore. The activity of this substrate is measured by determining the absorbance at 410 nm at 30 °C in 100 mM Tris / HCl buffer (pH 8.6) containing 0.1 wt% Brij® 35. In particular, the protease activity is determined using the method described in the examples. A suitable method for determining protease activity is also described in the prior art (Falkenberg F et al., “New robust subtilisins from halotolerant and halophilic Bacillaceae”, Appl Microbiol Biotechnol, 2023; Falkenberg F et al., “Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T”, FEBS Open Bio., 2022).

[0036] The protease can be added to the detergent composition as pure protein. However, the protease is typically used in the form of a stabilized, storable, and transportable preparation. Suitable preparations include granulated, extruded, or lyophilized compositions or highly concentrated aqueous solutions. In the case of an aqueous solution, polyhydric alcohols, such as glycerol or propanediol, are added to the protease for stabilization.

[0037] The preparation form preferably contains at least 1 wt.%, preferably 1 wt.% to 50 wt.%, particularly preferably 2 wt.% to 15 wt.%, most preferably 4 wt.% to 10 wt.% of active protease, based on the total mass of the protease preparation.

[0038] This preparation form is preferably added to the detergent composition in an amount of 0.1 wt.% to 10 wt.%, preferably 0.2 wt.% to 8 wt.%, most preferably 1 to 5 wt.%, in each case based on the total mass of the detergent composition.

[0039] Based on the active protease, the detergent composition preferably contains the protease in an amount of 0.0001 wt% to 5 wt%, preferably 0.001 wt% to 2.5 wt%, particularly preferably 0.005 wt% to 1 wt%, very particularly preferably 0.01 wt% to 0.5 wt%, in each case based on the total mass of active protease and the total mass of the detergent composition.

[0040] The detergent composition preferably comprises at least one surfactant as a detergent-active substance. The surfactant allows for effective removal of soils from the laundry to be cleaned. The surfactant can be selected, in particular, from the group of anionic, nonionic, cationic, and amphoteric surfactants. The detergent composition can comprise a single surfactant or a mixture of different surfactants.

[0041] The detergent composition preferably comprises at least one anionic or nonionic surfactant. More preferably, the detergent composition comprises both at least one anionic and at least one nonionic surfactant.

[0042] The total amount of surfactants in the detergent composition is preferably 10 wt% to 70 wt%, more preferably 20 wt% to 65 wt%, most preferably 40 wt% to 60 wt%, in each case based on the total mass of the detergent composition.

[0043] Anionic surfactants include, in particular, soaps, alkylbenzenesulfonates, alkanesulfonates, α-olefinsulfonates, α-sulfofatty acid esters, alkyl sulfates, alkenyl sulfates, alkyl ether sulfates, ether carboxylic acids and carboxylated alkylpolyglycosides.

[0044] The total amount of anionic surfactants is preferably 10 wt% to 70 wt%, more preferably 15 wt% to 60 wt%, most preferably 20 wt% to 40 wt%, in each case based on the total mass of the detergent composition.

[0045] Soap is the salt of a fatty acid. The fatty acid can be linear or branched, saturated or unsaturated. Preferably, a linear saturated or unsaturated fatty acid is used. A linear saturated fatty acid is particularly preferred.

[0046] The fatty acid preferably comprises at least 6 carbon atoms, more preferably at least 8 carbon atoms, most preferably at least 10 carbon atoms. The fatty acid particularly preferably comprises 6 to 32 carbon atoms, particularly preferably 8 to 28 carbon atoms, further preferably 10 to 18 carbon atoms, most preferably 12 to 18 carbon atoms. Preferred fatty acids are selected from the group consisting of caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and mixtures thereof. Lauric acid, myristic acid, palmitic acid, and stearic acid are particularly suitable.

[0047] Soaps exhibit particularly good properties in detergent compositions. The detergent composition therefore preferably comprises soaps in an amount of 1 wt.% to 20 wt.%, particularly preferably 3 wt.% to 15 wt.%, and most preferably 5 wt.% to 10 wt.%, based in each case on the total mass of the detergent composition.

[0048] Sulfuric acid semiesters of saturated or unsaturated fatty alcohols with 12 to 18 carbon atoms are preferably used as alkyl or alkenyl sulfates. Alkyl or alkenyl sulfates with 12 to 16 carbon atoms are preferred.

[0049] The alkyl ether sulfates used are preferably the sulfuric acid half esters of alkoxylated fatty alcohols. These are preferably ethoxylated or propoxylated fatty alcohols. The fatty alcohols preferably comprise 6 to 22 carbon atoms, particularly preferably 10 to 20 carbon atoms, most preferably 12 to 18 carbon atoms. The fatty alcohols are preferably alkoxylated with 1 to 20, particularly preferably 1 to 10, most preferably 2 to 8 repeating units. The alkyl ether sulfates particularly preferably have the following structural formula: R 1 -O-(R2-O) n -SO 3 - X + , where R 1 represents a linear or branched, saturated or unsaturated alkyl radical having 6 to 22 carbon atoms, particularly preferably 10 to 20 carbon atoms, most preferably 12 to 18 carbon atoms; R 2represents an alkenyl radical having 2 to 4 carbon atoms, particularly preferably 2 or 3 carbon atoms; n represents a number from 1 to 20, particularly preferably 1 to 10, most preferably 2 to 8; and X + for a cation, preferably an alkali metal cation, particularly preferably for K + or Na + stands.

[0050] The detergent composition preferably comprises alkyl ether sulfates in an amount of 1 wt% to 30 wt%, particularly preferably 5 wt% to 25 wt%, most preferably 10 wt% to 20 wt%, in each case based on the total mass of the detergent composition.

[0051] Compounds with the following structural formula are preferably used as alkylbenzenesulfonates: R 1 -Ph-SO 3 - X + , where R 1represents a linear or branched, saturated or unsaturated alkyl radical having 6 to 22 carbon atoms, particularly preferably 10 to 20 carbon atoms, most preferably 12 to 18 carbon atoms; Ph represents a phenyl radical; and X + for a cation, preferably an alkali metal cation, particularly preferably for K + or Na + stands.

[0052] A particularly preferred class of alkylbenzenesulfonates has the following structural formula: R 2 R 3 CH-Ph-SO 3 - X + , where R 2 represents a linear or branched, saturated or unsaturated alkyl radical having 6 to 22 carbon atoms, particularly preferably 8 to 20 carbon atoms, most preferably 12 to 18 carbon atoms; R 3represents a linear or branched, saturated or unsaturated alkyl radical having 1 to 10 carbon atoms, particularly preferably 1 to 5 carbon atoms, most preferably 1 to 3 carbon atoms; Ph represents a phenyl radical; and X + for a cation, preferably H + or an alkali metal cation, particularly preferably K + or Na + Preferably, the radicals R 2 and R 3 in this embodiment, a total of not more than 21 carbon atoms. Particularly preferably, R 2 and R 3 linear alkyl radicals.

[0053] Alkylbenzenesulfonates exhibit particularly good properties in detergent compositions. The detergent composition therefore preferably comprises alkylbenzenesulfonates in an amount of 7 wt.% to 40 wt.%, particularly preferably 10 wt.% to 30 wt.%, and most preferably 15 wt.% to 25 wt.%, based in each case on the total mass of the detergent composition.

[0054] Non-ionic surfactants are in particular alkoxylated fatty alcohols, such as fatty alcohol ethoxylates or propoxylates, alkoxylated fatty amines, alkoxylated alkanolamines, fatty acid amides, alkoxylated fatty acid amides, polyhydroxy fatty acid amides, alkylphenol polyglycol ethers, amine oxides, alkyl glucosides, alkyl polyglucosides and alkoxylated alkylphenols.

[0055] The total amount of nonionic surfactants is preferably 10 wt% to 70 wt%, more preferably 15 wt% to 60 wt%, most preferably 20 wt% to 40 wt%, in each case based on the total mass of the detergent composition.

[0056] Compounds with the following structural formula are preferably used as alkoxylated fatty alcohols: R1-O-(R2-O)nH, where R 1 represents a linear or branched, saturated or unsaturated alkyl radical having 6 to 22 carbon atoms, particularly preferably 10 to 20 carbon atoms, most preferably 12 to 18 carbon atoms; R 2 represents an alkenyl radical having 2 to 4 carbon atoms, particularly preferably 2 or 3 carbon atoms; and n represents a number from 1 to 20, particularly preferably 3 to 15, most preferably 5 to 10.

[0057] Alkoxylated fatty alcohols exhibit particularly good properties in detergent compositions. The detergent composition therefore preferably comprises alkoxylated fatty alcohols in an amount of 5 wt.% to 40 wt.%, particularly preferably 10 wt.% to 35 wt.%, and most preferably 15 wt.% to 30 wt.%, based in each case on the total mass of the detergent composition.

[0058] As alkoxylated alkanolamines, compounds with the following structural formula are preferably used: (HO-R 1 -O-(R 2 -O)nR 3 )3-N where the residues R 1 , R 2 , R 3each independently represents an alkenyl radical having 2 to 4 carbon atoms, and n represents a number from 5 to 30, particularly preferably 10 to 25, most preferably 15 to 20. A particularly preferred alkoxylated amine is propoxylated and ethoxylated triethanolamine. Such an amine is available, for example, under the name Plurafac® LF 1430 from BASF.

[0059] Alkoxylated alkanolamines exhibit particularly good properties in detergent compositions. The detergent composition therefore preferably comprises alkoxylated alkanolamines in an amount of 1 wt.% to 20 wt.%, particularly preferably 2 wt.% to 15 wt.%, and most preferably 3 wt.% to 10 wt.%, based in each case on the total mass of the detergent composition.

[0060] In a preferred embodiment, the detergent composition comprises an alkylbenzenesulfonic acid, a fatty acid, an alkoxylated fatty alcohol and an alkoxylated alkanolamine.

[0061] Amphoteric surfactants include, in particular, betaines, sulfobetaines and imidazoline derivatives such as cocoamphodiacetate.

[0062] The total amount of amphoteric surfactants in the detergent composition is preferably 0.1 wt% to 20 wt%, particularly preferably 1 wt% to 15 wt%, further preferably 2 wt% to 10 wt%, most preferably 3 wt% to 7 wt%, in each case based on the total mass of the detergent composition.

[0063] In one embodiment, the detergent composition does not comprise amphoteric surfactants.

[0064] Cationic surfactants are in particular fatty amine salts and quaternary ammonium compounds.

[0065] However, the detergent composition preferably comprises no or only a limited amount of cationic surfactants. Accordingly, the detergent composition preferably comprises no more than 10% by weight, particularly preferably no more than 1% by weight, and most preferably no more than 0.1% by weight of cationic surfactants, in each case based on the total mass of the detergent composition.

[0066] In a particularly preferred embodiment, the detergent composition does not comprise any cationic surfactants.

[0067] The detergent composition may further comprise at least one complexing agent. Complexing agents are compounds that bind metal ions, particularly alkaline earth metal ions such as magnesium or calcium ions.

[0068] The total amount of complexing agents in the detergent composition is preferably 0.1 wt% to 5 wt%, preferably 0.25 wt% to 3 wt%, most preferably 0.5 wt% to 2 wt%, in each case based on the total mass of the detergent composition.

[0069] Complexing agents are in particular phosphates, such as diphosphate and triphosphate salts, phosphonic acids, such as 1-hydroxyethene-1,1-diphosphonic acid, amino-tri(methylenephosphonic acid) and diethylenetriaminepentakis(methylenephosphonic acid) (DTPMP), carboxylic acids, in particular di- or polycarboxylic acids, such as citric acid, nitrilotriacetic acid (NTA), N-(2-hydroxyethyl)-iminodiacetic acid, ethylenediaminetriacetic acid (EDTA), 1,2,3,4-cyclopentanetetracarboxylic acid, L-glutamic acid-N,N-diacetic acid (GLDA) and methylglycinediacetic acid (MGDA), and ion exchangers, such as polyacrylic acid, poly-(α-hydroxyacrylic acid), poly-[(3-hydroxymethyl)-hexamethylene-1,3,5-tricarboxylic acid], Poly-[(4-methoxy)-tetramethylene-1,2-dicarboxylic acid], poly-(tetramethylene-1,2-dicarboxylic acid), and silicates.

[0070] In a preferred embodiment, the detergent composition comprises complexing agents selected from the group of phosphonic acids in an amount of 0.1 wt.% to 5 wt.%, preferably 0.25 wt.% to 3 wt.%, most preferably 0.5 wt.% to 2 wt.%, in each case based on the total mass of the detergent composition.

[0071] In a further embodiment, the amount of phosphonates in the detergent composition is limited to 2% by weight or less, preferably 1% by weight or less, more preferably 0.1% by weight or less. In a particularly preferred embodiment, the detergent composition does not comprise any phosphonates.

[0072] In a particularly preferred embodiment, the detergent composition comprises complexing agents selected from the group consisting of 1-hydroxyethene-1,1-diphosphonic acid, aminotri(methylenephosphonic acid) and diethylenetriaminepentakis(methylenephosphonic acid) (DTPMP) in an amount of 0.1 wt% to 5 wt%, preferably 0.25 wt% to 3 wt%, most preferably 0.5 wt% to 2 wt%, in each case based on the total mass of the detergent composition.

[0073] In a preferred embodiment, the detergent composition comprises at least one di- or polycarboxylic acid as a complexing agent. In a particularly preferred embodiment, the detergent composition comprises citric acid, 1,2,3,4-cyclopentanetetracarboxylic acid, L-glutamic acid-N,N-diacetic acid (GLDA), methylglycinediacetic acid (MGDA), or mixtures thereof as a complexing agent.

[0074] In one embodiment, the detergent composition comprises complexing agents selected from the group consisting of di- or polycarboxylic acids in an amount of preferably 0.1 wt.% to 5 wt.%, preferably 0.25 wt.% to 3 wt.%, most preferably 0.5 wt.% to 2 wt.%, in each case based on the total mass of the detergent composition.

[0075] In one embodiment, the detergent composition comprises complexing agents selected from the group consisting of citric acid, 1,2,3,4-cyclopentanetetracarboxylic acid, L-glutamic acid-N,N-diacetic acid (GLDA), methylglycinediacetic acid (MGDA), or mixtures thereof in an amount of preferably 0.1 wt% to 5 wt%, more preferably 0.25 wt% to 3 wt%, most preferably 0.5 wt% to 2 wt%, in each case based on the total mass of the detergent composition.

[0076] The detergent composition further comprises water in a preferred amount of up to 50% by weight, based on the total mass of the detergent composition. Preferably, the detergent composition comprises up to 40% by weight of water, more preferably up to 30% by weight of water, most preferably up to 15% by weight of water, each based on the total mass of the detergent composition.

[0077] The detergent composition may further comprise at least one other solvent besides water. Other solvents include, in particular, mono- or polyhydric alcohols, alkanolamines, and glycol ethers.

[0078] The amount of the further solvent is preferably 5 wt% to 50 wt%, preferably 8 wt% to 30 wt%, most preferably 10 wt% to 20 wt%, in each case based on the total mass of the detergent composition.

[0079] The detergent composition is preferably liquid. The total amount of water and other solvents is therefore preferably 5% by weight or more, more preferably 10% by weight or more, most preferably 15% by weight or more.

[0080] In a preferred embodiment, the detergent composition comprises at least one polyhydric alcohol (polyol). Suitable polyols are, in particular, those alcohols having two or more OH groups and 2 to 8 carbon atoms. Polyols having 2 or 3 OH groups and 3 to 6 carbon atoms are particularly suitable. Suitable polyols include, in particular, propanediol, in particular 1,2-propanediol; butanediol, in particular 1,2-butanediol, 1,3-butanediol, and 1,4-butanediol; glycerol, or mixtures thereof.

[0081] The total amount of polyols is preferably 5 wt% to 50 wt%, preferably 8 wt% to 30 wt%, most preferably 10 wt% to 20 wt%, in each case based on the total mass of the detergent composition.

[0082] The total amount of polyols having two or more OH groups and 2 to 8 carbon atoms is preferably 5 wt% to 50 wt%, preferably 8 wt% to 30 wt%, most preferably 10 wt% to 20 wt%, in each case based on the total mass of the detergent composition.

[0083] In a preferred embodiment, the detergent composition comprises propanediol in an amount of 1 wt% to 20 wt%, preferably 2 wt% to 8 wt%, and glycerol in an amount of 1 wt% to 20 wt%, preferably 5 wt% to 15 wt%, each based on the total mass of the detergent composition.

[0084] The detergent composition may further comprise one or more adjuvants. These include, in particular, bleaching agents, such as hypochlorite bleach, perborates, percarbonates, perphosphates, and percarbamides; whiteners; enzyme stabilizers, foam inhibitors, corrosion inhibitors, dyes, fragrances, and preservatives.

[0085] The excipients include, in particular, polymeric enhancers such as polyethyleneimines and propylene glycol terephthalates.

[0086] The detergent composition may also contain other enzymes besides the protease described above. For example, the detergent composition may contain amylases, cellulases, dispersins, lipases, tannases, mannases, and polyesterases. In the case of enzymes obtained through fermentation, these can be used together with ingredients of the fermentation medium.

[0087] In a preferred embodiment, however, the detergent composition does not comprise any further enzymes apart from the protease described above in order to prevent proteolytic degradation of these further enzymes. It has proven extremely advantageous to combine any further enzymes with the detergent composition according to the invention only immediately before use in order to ensure maximum activity of the further enzymes. In this case, the detergent composition can be used in particular as one of several compositions in a multi-chamber system. In this case, the detergent composition according to the invention is accommodated in one of the chambers, while the remaining chambers each contain one or more compositions with the further enzymes. When the detergent composition is used, the chambers are opened in order to combine the individual compositions into a detergent.

[0088] The detergent composition preferably has a pH of 7 to 12, more preferably 7.5 to 9, most preferably 7.5 to 8.5. The pH is determined at 20°C. If the detergent composition has too low a water content to reliably determine the pH, the composition is diluted with water for the measurement. The pH is preferably adjusted by adding a suitable base. The detergent composition preferably comprises a base selected from the group consisting of amines, alkali metal carbonate, and alkali metal hydroxide, more preferably selected from monoethanolamine, sodium carbonate, and sodium hydroxide.

[0089] Conventional detergents usually contain boric acid or boric acid salts (borates) to stabilize enzymes. However, according to the present invention, the use of boric acid or borates is not absolutely necessary to stabilize the protease. Rather, the invention makes it possible to dispense with the addition of boric acid or borates. In one embodiment, the amount of boric acid and borates in the detergent composition is therefore limited to no more than 1% by weight, preferably no more than 0.5% by weight, more preferably no more than 0.1% by weight, most preferably no more than 0.01% by weight, in each case based on the total mass of the detergent composition. In a particularly preferred embodiment, the detergent composition does not comprise boric acid or borates.

[0090] A further aspect of the invention relates to a process for producing a detergent composition having improved storage stability by adding the protease described above to the detergent composition described above.

[0091] The process is characterized in particular in that the activity of the protease in the detergent composition after storage of the detergent composition at a temperature of 40 °C for a period of two weeks, preferably four weeks, is at least 90%, preferably at least 95%, further preferably at least 98%, particularly preferably at least 99% of the activity of the protease immediately after preparation of the detergent composition.

[0092] A further aspect of the invention also relates to the use of the above-described protease for producing a detergent composition described above with the purpose of increasing the storage stability of the detergent composition. In particular, the use serves the purpose of ensuring that the activity of the protease in the detergent composition, after storage of the detergent composition at a temperature of 40°C for a period of two weeks, preferably four weeks, is at least 90%, preferably at least 95%, further preferably at least 98%, particularly preferably at least 99% of the activity of the protease immediately after production of the detergent composition.

[0093] A further aspect of the invention relates to a method for cleaning laundry, in particular for cleaning dishes or textiles, by contacting the laundry with a detergent composition described above, characterized in that the detergent composition contains a protease described above. Preferably, the detergent composition is diluted with water before contacting it with the laundry. Preferably, the detergent composition is contacted with the laundry at a temperature of 10°C to 90°C, preferably 15°C to 60°C, particularly preferably 20°C to 40°C.

[0094] A further aspect of the invention relates to the use of the protease described above in a detergent composition described above for cleaning laundry, in particular for cleaning dishes or textiles. Examples

[0095] The present invention will now be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

[0096] The storage stability of a protease according to SEQ ID NO: 1 was determined in a detergent composition with Bacillus subtilis culture supernatants containing the protease SEQ ID NO: 1. The culture supernatants were diluted 1:4 with a detergent matrix (1 part by weight of culture supernatant to 3 parts by weight of the detergent matrix). The resulting detergent composition was stored at a temperature of 30°C or 40°C for eight weeks. The protease activity was measured immediately after preparation of the detergent composition and after two, four, and eight weeks. In parallel, a detergent composition containing the alkaline protease Savinase (PDB number 1SVN; UniProtKB number P29600) was prepared, and the activity of Savinase® was determined in the same way. Savinase has less than 60% sequence identity to SEQ ID NO: 1.

[0097] Protease activity was determined by the release of the chromophore para-nitroaniline from the substrate succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; Bachem L-1400). The release of the chromophore causes an increase in absorbance at 410 nm, the time course of which is a measure of enzymatic activity. The measurement was carried out at a temperature of 30 °C, pH 8.6, and a wavelength of 410 nm. The measurement time was 5 min with a measurement interval of 20 to 60 seconds. The measurement was carried out in a solution consisting of 10 µl AAPFpNA solution (70 mg / ml), 10 µl protease solution, and 1000 µl Tris / HCl (0.1 M, pH 8.6 with 0.1% Brij 35).

[0098] The composition of the detergent matrix is ​​given in the following table: component Wt.% Alkylbenzenesulfonic acid 22 Fatty acid C12-C18 7 Ethoxylated fatty alcohol C12-C18, 7 EO 20 Alkoxylated alkanolamine 5 1,2-Propanediol 4 Glycerol 10 Monoethanolamine 6 DTPMP 1 Ethoxylated polyethyleneimine 6 Propylene glycol terephthalate 2 Other additives 1 5,1 Water rest 1 Bitter substances, bleaching agents, preservatives, dyes, fragrances

[0099] The determined protease activities are shown in the following table, where the activity is given in relation to the activity measured immediately after production. enzyme temperature 2 weeks 4 weeks 8 weeks Savinase 30 °C 91% 86% 74% SEQ ID NO: 1 30 °C 100% 100% 100% Savinase 40 °C 63% 53% 20% SEQ ID NO: 1 40 °C 100% 100% 95%

[0100] These results demonstrate that a protease according to SEQ ID NO: 1 according to the invention in a detergent composition exhibits an activity of at least 90% relative to the original activity when the detergent composition was prepared, even after eight weeks of storage at a temperature of up to 40°C. In comparison, the activity of an alternative protease decreases after just two weeks of storage and no longer reaches 90% of the initial activity after just four weeks of storage. A detergent composition according to the invention containing a protease according to SEQ ID NO: 1 therefore exhibits improved storage stability. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited non-patent literature

[0000] Madeira F, Pearce M, Tivey ARN, et al. “Search and sequence analysis tools services from EMBL-EBI in 2022”, Nucleic Acids Research, 2022

[0030] Falkenberg F et al., “New robust subtilisins from halotolerant and halophilic Bacillaceae”, Appl Microbiol Biotechnol, 2023 [0031, 0035] Falkenberg F et al., “Biochemical characterization of a novel oxidatively stable, halotolerant, and high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T”, FEBS Open Bio., 2022 [0031, 0035]

Claims

[1] Detergent composition comprising a protease, characterized by that the amino acid sequence of the protease has an identity of 60% or more to one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3. [2] Detergent composition according to claim 1, wherein the activity of the protease in the detergent composition after storage of the detergent composition at a temperature of 40°C for a period of two weeks is at least 90% of the activity of the protease immediately after preparation of the detergent composition. [3] A detergent composition according to claim 1 or 2, wherein the detergent composition contains 0.0001 wt% to 5 wt% of the protease, based on the total mass of active protease and the total mass of the detergent composition. [4] Detergent composition according to any one of claims 1 to 3, wherein the detergent composition has a pH in the range of 7.5 to 9. [5] Detergent composition according to any one of claims 1 to 4, wherein the detergent composition 10 wt% to 70 wt% anionic and / or non-ionic surfactants, 5 wt% to 50 wt% polyols, 0.1 wt% to 5 wt% complexing agent, and up to 50 wt% water. [6] Detergent composition according to any one of claims 1 to 5, wherein the detergent composition comprises at least one polyol having 3 to 6 carbon atoms and 2 to 3 OH groups. [7] Detergent composition according to any one of claims 1 to 6, wherein the detergent composition comprises at least one alkyl ether sulfate and / or at least one alkoxylated fatty alcohol. [8] Detergent composition according to any one of claims 1 to 7, wherein the detergent composition comprises not more than 1% by weight of boric acid and borates, based on the total mass of the detergent composition. [9] A process for preparing a detergent composition with improved storage stability by adding a protease to the detergent composition, characterized by that the amino acid sequence of the protease has an identity of 60% or more to one of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3. [10] A method for cleaning laundry, in particular for cleaning dishes or textiles, by bringing the laundry into contact with a detergent composition according to claim 1.