Automatic dishwashing compositions comprising a protease

Abstract

The present invention relates to fabric care and home care compositions comprising a surfactant and a protease.

Description

[Technical field]

[0001] The present invention is in the field of automatic dishwashing compositions. [Background technology]

[0002] Proteases (also known as proteinases) are enzymes that have the ability to degrade other proteins. Proteases have the ability to perform proteolysis, which initiates protein catabolism by hydrolysis of peptide bonds that link amino acids together in the peptide or polypeptide chain that forms the protein. This activity of proteases as protein-digesting enzymes is called proteolytic activity. There are many well-known procedures for measuring proteolytic activity (Kalisz, "Microbial Proteinases," in Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology, (1988)). For example, proteolytic activity can be confirmed by comparative assays that analyze the ability of each protease to hydrolyze a commercially available substrate. Exemplary substrates useful in the analysis of protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and Keratin Azure (Sigma-Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (see, for example, WO 99 / 34011 and US Pat. No. 6,376,450, both of which are incorporated herein by reference).

[0003] Serine proteases are enzymes (EC number 3.4.21) that have an active site serine that initiates the hydrolysis of peptide bonds in proteins. Serine proteases comprise a diverse class of enzymes with a wide range of specificities and biological functions, which are further classified as chymotrypsin-like (trypsin-like) and subtilisin-like based on their structure. The prototypical subtilisin (EC number 3.4.21.62) was first obtained from Bacillus subtilis. Subtilisins and their homologs are members of the S8 peptidase family of the MEROPS classification scheme (Rawlings, ND et al (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 44, D343-D350). Members of family S8 have a catalytic triad in their amino acid sequence in the order Asp, His, and Ser. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 99 / 34011 [Patent Document 2] U.S. Patent No. 6,376,450 [Non-patent literature]

[0005] [Non-Patent Document 1] Kalisz, "Microbial Proteinases," in Advances in Biochemical Engineering / Biotechnology, (1988), edited by Fiechter. Summary of the Invention [Problem to be solved by the invention]

[0006] Although many useful variant proteases have been developed for cleaning applications, there remains a need for improved protease variants.

[0007] There also remains a need for improved protease variants that have improved stability during manufacture of the variants, during storage of compositions containing the variants, and during use (e.g., in wash baths), particularly in oxidizing environments, including when the variants are used in combination with bleach.

[0008] There also remains a need to provide automatic dishwashing detergent compositions that provide good cleaning and can have good stability.

[0009] Additionally, there remains a need to provide an automatic dishwashing composition in the form of a water soluble unit dose pouch containing a compartment, the compartment containing both a powder component and a gel component, the composition containing a surfactant and a protease, the composition having good cleaning performance, good protease performance, and good stability. [Means for solving the problem]

[0010] The present invention provides an automatic dishwashing composition comprising a surfactant and a protease, The automatic dishwashing composition is in the form of a water-soluble unit dose pouch containing a compartment, the compartment containing both a powder component and a gel component; The protease has the following sequence with respect to SEQ ID NO:1: (i) a mutation at position 122, (ii) one or more mutations at positions 126, 127, 128, 211, and 212; The present invention relates to an automatic dishwashing composition, wherein the subtilisin variant comprises DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Automatic dishwashing compositions The automatic dishwashing composition comprises a surfactant and a protease.

[0012] The automatic dishwashing composition is in the form of a water-soluble unit dose pouch containing a compartment, the compartment containing both a powder component and a gel component.

[0013] Preferably, the composition is in the form of a multi-compartment pouch containing two or more compartments, preferably three or more compartments, at least one of the compartments containing both a powder component and a gel component.

[0014] The protease may be present in the gel component.

[0015] The composition may include a bleaching agent, which is present in the powder component.

[0016] The compositions of the present invention are very well suited to be provided in the form of a multi-compartment pack, more particularly in a multi-compartment pack comprising compartments with compositions in different physical forms, for example a compartment containing the composition in solid form and another compartment containing the composition in liquid form. The compositions are preferably encapsulated in a water-soluble film such as polyvinyl alcohol. Particularly preferred are compositions in unit dose form wrapped in a polyvinyl alcohol film having a thickness of less than 100 μm, preferably 20-90 μm. The detergent compositions of the present invention have a weight of about 8 to about 25 grams, preferably about 10 to about 20 grams. This weight range fits comfortably into the dispenser of a dishwasher. Although this range corresponds to a small amount of detergent, the detergent is formulated to provide all of the benefits as described herein above.

[0017] The composition is preferably phosphate-free. By "phosphate-free" herein is understood the composition containing less than 1% phosphate by weight of the composition, preferably less than 0.1% phosphate.

[0018] The composition is typically a detergent composition. "Detergent composition" or "detergent formulation" is used in reference to a composition intended for use in a washing medium to clean soiled or unclean objects. In some embodiments, the detergent of the present disclosure comprises one or more subtilisin variants described herein, and additionally one or more surfactants, transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, fluorescent dyes, anti-caking agents, masking agents, enzyme stabilizers, calcium, enzyme activators, antioxidants, and / or solubilizers. In some cases, the builder salts are a mixture of silicates and phosphates, preferably with more silicates (e.g., sodium metasilicate) than phosphates (e.g., sodium tripolyphosphate). Some embodiments relate to cleaning or detergent compositions that do not contain any phosphates (e.g., phosphates or phosphate builders).

[0019] The term "auxiliary material" refers to any liquid, solid, or gaseous material contained in a cleaning composition other than one or more subtilisin variants described herein, or recombinant polypeptides or active fragments thereof. In some embodiments, the cleaning compositions of the present disclosure include one or more cleaning auxiliary materials. Each cleaning auxiliary material is typically selected depending on the particular type and form of the cleaning composition (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel, foam, or other composition). Preferably, each cleaning auxiliary material is compatible with the protease enzyme used in the composition.

[0020] The phrases "substantially boron-free composition" or "substantially boron-free detergent" refer to a composition or detergent, respectively, that contains trace amounts of boron, e.g., less than about 1000 ppm (1 mg / kg or liter equals 1 ppm), less than about 100 ppm, less than about 50 ppm, less than about 10 ppm, or less than about 5 ppm, or less than about 1 ppm, possibly from other composition or detergent ingredients.

[0021] The term "bleaching" refers to treating a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient length of time and / or under appropriate pH and / or temperature conditions to brighten (i.e., whiten) and / or clean the material. Examples of chemicals suitable for bleaching include, but are not limited to, ClO2, H2O2, peracid, NO2, etc. Bleaching agents also include enzymatic bleaches, such as perhydrolases and arylesterases. Another embodiment relates to a composition comprising one or more subtilisin variants described herein and one or more perhydrolases, such as those described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0022] The term "cleaning performance" of a protease (e.g., one or more subtilisin variants described herein, or a recombinant polypeptide or active fragment thereof) refers to the cleaning contribution of one or more subtilisin variants described herein that provide additional cleaning performance to a detergent compared to a detergent that does not have one or more subtilisin variants described herein added to the composition. The cleaning performance is compared under relevant cleaning conditions. In some test systems, other relevant factors such as detergent composition, foam concentration, water hardness, cleaning mechanism, time, pH, and / or temperature can be controlled to mimic conditions typical of domestic use in a particular market segment (e.g., automatic dishwashing, dishwashing, tableware washing, etc.).

[0023] The phrase "relevant washing conditions" is used herein to indicate the conditions actually used in households in the hand dishwashing, automatic dishwashing or laundry detergent market segments, in particular washing temperature, time, washing mechanism, foam concentration, detergent type and water hardness.

[0024] The term "dishwashing" refers to both domestic and industrial dishwashing, and relates to both automatic dishwashing (eg, in a dishwashing machine).

[0025] The term "disinfecting" refers to the removal of contaminants from a surface, as well as the inhibition or killing of microorganisms on the surface of an article.

[0026] The term "compact" form of the cleaning compositions herein is best reflected by the density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional components of detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically about 17 to about 35% by weight of the total composition. In contrast, in compact compositions, the filler salts are present in an amount not exceeding about 15% by weight of the total composition. In some embodiments, the filler salts are present in an amount not exceeding about 10% by weight of the composition, or more preferably about 5% by weight. In some embodiments, the inorganic filler salts are selected from alkali metal and alkaline earth metal salts of sulfates and chlorides. In some embodiments, the filler salt is sodium sulfate.

[0027] Gel Ingredients The gel component may also be referred to herein as a gel, a gel-like phase, or a gel phase.

[0028] The gel component is preferably anhydrous, meaning containing less than 10%, or less than 8%, or less than 6%, or less than 4%, or less than 2%, or less than 1% water by weight, or containing no intentionally added water.

[0029] The gel component typically includes enzymes, typically including a protease, and if present, typically also including an amylase.

[0030] The gel component may comprise an enzyme selected from a protease, an amylase, a lipase, a hemicellulase, a cellulase, a perhydrolase or an oxidoreductase, and combinations thereof.

[0031] Typically, a gel component should be understood to mean a composition / phase with an internal structured network. This internal structured (spatial) network is formed by dispersing solid but distributed substances and long or highly branched particles and / or gelling agents in at least one liquid (at least one liquid being liquid at 20° C.). Such gel phases behave thermoreversibly.

[0032] This gel phase may be, for example, fluid or dimensionally stable. However, according to the invention, the gel-like phase is preferably dimensionally stable at room temperature. In the preparation, a gel-forming agent, preferably xanthan, gelatin or polyvinyl alcohol and / or derivatives thereof, is contacted with a solvent, preferably an organic solvent, preferably one or more polyhydric alcohols. This results in a fluid mixture that can be brought into the desired shape. After a certain time, a gel phase is obtained that remains in the given shape, i.e. is dimensionally stable. This time, i.e. the solidification time, is preferably not more than 15 minutes, preferably not more than 10 minutes, particularly preferably not more than 5 minutes. At the same time, the at least one gel phase yields to pressure but does not deform as a result and returns to its initial state after the pressure has ceased. The at least one gel phase is preferably elastic, in particular linearly elastic.

[0033] At least one gel phase is preferably a molded body. A molded body is a unitary body that stabilizes itself in the applied shape. This dimensionally stable body is formed from a molding material (e.g., composition) by forming this molding material into a predetermined shape in a targeted manner, for example by pouring a liquid composition into a mold and then curing the liquid composition, for example within the scope of a sol-gel process. Certain minimum requirements are imposed on the formulation of at least one gel phase. Thus, as already mentioned, the gel phase must solidify in the shortest possible time. Longer solidification times lead to longer manufacturing times and thus higher costs. According to the present invention, solidification time means the period during which at least one gel phase changes during preparation from a free-flowing state to a dimensionally stable state that is not free-flowing at room temperature. It is at a temperature below room temperature, typically 20°C.

[0034] At least one gel phase is preferably a solid gel phase, in which case it is cut-resistant: it can be cut, for example, with a knife after solidification, without being further destroyed apart from the cut made.

[0035] In addition, at least one gel phase is preferably translucent or transparent, resulting in a good optical impression. The transmittance of the gel phase (without dye) is preferably in the range of 100% to 20%, 100% to 30%, in particular 100% to 40%. To measure the light transmittance (transmittance), the transmittance (%) at 600 nm was determined against water as a reference at 20°C. For this purpose, the composition was poured into a prepared 11 mm round cuvette and measured according to length with a LICO 300 colorimetric system after a storage time of 12 hours at room temperature.

[0036] At least one gel phase is low in water. For the purposes of the present invention, low water content means that a small amount of water can be used to prepare at least one gel phase. The proportion of water in the gel phase is in particular below 20% by weight, preferably below 15% by weight, in particular below 12% by weight, in particular from 10 to 5% by weight. The data in weight percent are based on the total weight of the gel phase. This has the advantage that a small amount of water in combination with PVOH can have a structure-forming or gel-forming effect.

[0037] According to a preferred embodiment, at least one gel phase is essentially anhydrous. This means that the gel phase is preferably essentially free of water. "Substantially free" here means that small amounts of water may be present in the gel phase. This water can be introduced into the phase, for example, by a solvent or as water of crystallization or by reaction between the components of the phase. However, since a solvent is used for the preparation of the gel phase, only small amounts, in particular no water, are preferred. In this embodiment, the proportion of water in the gel phase is 4.9% by weight or less, 4% by weight or less, preferably 2% by weight or less, in particular 1% by weight or less, in particular 0.5% by weight or less, in particular 0.1% by weight or less or 0.05% by weight or less. The weight percentages are based on the total weight of the gel phase.

[0038] The gel component typically comprises a gel former, preferably selected from gelatin, xanthan and / or polyvinyl alcohol, in particular gelatin or polyvinyl alcohol, particularly preferably polyvinyl alcohol, in an amount of in each case 4 to 40% by weight, in particular 6 to 30% by weight, particularly preferably 7 to 24% by weight, very particularly preferably 8 to 22% by weight, in particular for example 14 to 20% by weight, based on the total weight of the gel-like phase.

[0039] Preferably, at least one gel phase contains, particularly preferably, PVOH (polyvinyl alcohol) and / or its derivatives. Polyvinyl alcohol is a thermoplastic material that is usually prepared as a white to yellowish powder by hydrolysis of polyvinyl acetate. Polyvinyl alcohol (PVOH) is resistant to almost all anhydrous organic compounds.

[0040] Polyvinyl alcohols with a molar mass between 30,000 and 60,000 g / mol are preferred.

[0041] The preferred derivatives of PVOH for the purposes of the present invention are copolymers of polyvinyl alcohol with other monomers, in particular copolymers with anionic monomers.Suitable anionic monomers are preferably vinyl acetate, alkyl acrylates, maleic acid and its derivatives, in particular monoalkyl maleates (in particular monomethyl maleate), dialkyl maleates (in particular dimethyl maleate), maleic anhydride, fumaric acid and its derivatives, in particular monoalkyl fumarate (in particular monomethyl fumarate), dialkyl fumarate (in particular dimethyl fumarate), fumaric anhydride, itaconic acid and its derivatives, in particular dialkyl itaconate, dimethyl itaconate, itaconic anhydride, citraconic acid (methyl maleate) and its derivatives, monoalkyl citraconic acid (in particular methyl citraconic acid), monoalkyl citraconic acid (in particular methyl citraconic acid), Dialkylcitraconates (dimethylcitraconate), citraconic anhydride, mesaconic acid (methyl fumaric acid) and its derivatives, monoalkyl mesaconic acid, dialkyl mesaconic acid, mesaconic anhydride, glutaconic acid and its derivatives, monoalkyl glutaconic acid, dialkyl glutaconic acid, glutaconic anhydride, vinylsulfonic acid, alkylsulfonic acid, ethylenesulfonic acid, 2-acrylamido-1-methylpropanesulfonic acid, 2-acrylamido-2-methylpropanesulfonic acid, 2-methylacrylamido-2-methylpropanesulfonic acid, 2-sulfoethyl acrylate, and combinations thereof, as well as the alkali metal salts or esters of the above-mentioned monomers. Particularly preferred derivatives of PVOH are those selected from copolymers of polyvinyl alcohol with monomers, in particular those selected from the group of monoalkyl maleates (especially monomethyl maleate), dialkyl maleates (especially dimethyl maleate), maleic anhydride, and combinations thereof, as well as the alkali metal salts or esters of the above-mentioned monomers. The values ​​given for the polyvinyl alcohol itself apply to the preferred molecular weights. For the purposes of the present invention, it is preferred that at least one gel phase comprises by weight polyvinyl alcohol and / or its derivatives, preferably polyvinyl alcohol having a degree of hydrolysis preferably between 70 and 100 mol %, in particular between 80 and 90 mol %, particularly preferably between 81 and 89 mol %, in particular between 82 and 88 mol %.

[0042] Particularly preferred is polyvinyl alcohol as a white-yellowish powder or granules having a degree of polymerization in the range of about 100 to 2500 (molar mass of about 4000 to 100,000 g / mol) and a degree of hydrolysis of 80 to 99 mol%, preferably 85 to 90 mol%, in particular 87 to 89 mol%, for example 88 mol%, and correspondingly containing a residual content of acetyl groups.

[0043] PVOH powders having the above mentioned properties suitable for use in at least one gel phase are sold, for example, by Kuraray under the name Mowiol® or Poval®. Also suitable, for example, is Exceval® AQ4104 from Kuraray. Particularly suitable are Mowiol C30 from Kuraray, Poval® qualities, in particular qualities 3-83, 3-88, 6-88, 4-85, particularly preferably 4-88, very particularly preferably Poval 4-88 S2, and Mowiol® 4-88.

[0044] The water solubility of polyvinyl alcohol can be determined by post-treatment with aldehydes (acetalization) or ketones (ketalization). Polyvinyl alcohols that are acetalized or ketalized with aldehyde or keto groups of sugars or polysaccharides or mixtures thereof have proven to be particularly preferred and particularly advantageous due to their very good solubility in cold water. The reaction products of polyvinyl alcohol and starch can be used very advantageously. Furthermore, the solubility in water can be changed by complexing with Ni or Cu salts or by treatment with dichromate, boric acid, borax, and thus adjusted to the desired value in a targeted manner.

[0045] According to a preferred embodiment, the at least one enzyme or enzymes, preferably the protease variant of the invention, are not introduced into the gel phase as a liquid enzyme preparation, in particular into a gel phase with a gel former. It is particularly preferred that the liquid enzyme-enzyme preparation is not introduced into a gel phase in which the gel former is selected from gelatin, xanthan and / or polyvinyl alcohol, in particular gelatin or polyvinyl alcohol, particularly preferably polyvinyl alcohol. In particular in the preparation of a PVOH-containing gel phase, the introduction of the liquid enzyme preparation into the gel phase, which remains heated by the preparation, is carried out at high temperature, which leads to a considerable decrease in the stability of the enzyme in the liquid enzyme preparation. The decrease in stability means that the activity of the enzyme is significantly decreased compared to the starting preparation. This decrease in stability can be measured, for example, by measuring the residual enzyme activity or by comparing the purification performance of the corresponding enzyme, in particular according to the IHF standard. The enzyme is particularly preferably incorporated into the gel phase as granules.

[0046] PVOH is particularly suitable for preparing a gel phase that meets the above requirements.Therefore, in addition to at least one enzyme, particularly preferably at least one enzyme granule selected from the group of proteases and / or amylases, PVOH and at least one polyhydric alcohol, at least one gel phase is particularly preferred.Particularly preferably, the at least one gel phase comprises at least one amylase granule and / or at least one protease granule, PVOH and at least one polyhydric alcohol.

[0047] According to the invention, the at least one gel phase preferably comprises at least one enzyme, preferably at least one enzyme granule, preferably at least one amylase granule and / or at least one protease granule, in particular at least one protease granule, PVOH and / or its derivatives in a proportion of about 4% to 40% by weight, in particular 6% to 30% by weight, preferably 7 to 24% by weight, particularly preferably 8% to 22% by weight. A significantly lower proportion of PVOH does not result in the formation of a stable gel phase. The values ​​are each based on the total weight of the gel phase.

[0048] These gel phases thus prepared have a particularly high melting point, are dimensionally stable (even at 40° C.) and do not change their shape, or change their shape only slightly, even during storage. In particular, they are also less reactive with respect to direct negative interactions with the components of the granular mixture.

[0049] In particular, PVOH can also produce gel phases without difficulty that contain little or no water content. When PVOH is used as the polymer for at least one gel phase, a low-viscosity melt results at 110-120°C, which can therefore be processed particularly easily. In particular, the gel phases can be rapidly and accurately introduced into the water-soluble coating without sticking occurring or the amounts being inaccurately metered. Furthermore, these gel phases adhere particularly well to the water-soluble coating, especially if it is also produced from PVOH.

[0050] This is also optically advantageous. As a result of the rapid solidification of at least one gel phase with PVOH, the further processing of the gel phase can be carried out particularly quickly. Furthermore, the good solubility of the gel phase produced is particularly favorable for the overall solubility of the detergent. In addition, a gel phase with such a short solidification time is advantageous because the at least one solid phase containing a granular mixture, especially a powder, introduced thereon does not sink into the gel that is not yet completely solidified or is too soft. This results in a visually unpleasant detergent part.

[0051] In particular, in the case of multi-phase disposable portions according to the invention having at least one solid phase, it is important that at least one gel phase is dimensionally stable, so that possible interactions between the solid and the gel phase are as minimal as possible. If at least one gel phase contains gelatin in addition to PVOH, the toughness of the gel phase in the preparation is surprisingly increased.

[0052] The present invention further preferably provides a cleaning agent, preferably a dishwashing agent, in particular an automatic dishwashing agent, comprising in addition to at least one enzyme, preferably at least one enzyme granule, at least one organic solvent, in particular selected from 1,2-propanediol, 1,3-propanediol, glycerol, 1,1,1-trimethylolpropane, triethylene glycol, dipropylene glycol, polyethylene glycol and / or mixtures thereof.

[0053] The at least one gel phase preferably comprises at least one polyhydric alcohol. In addition to the production of a fluid gel phase, the at least one polyhydric alcohol also allows the preparation of a dimensionally stable non-fluid gel phase within a short setting time of less than 15 minutes, in particular less than 10 minutes. Polyhydric alcohols for the purposes of the present invention are hydrocarbons in which two, three or more hydrogen atoms are replaced by OH groups. The OH groups are in each case bonded to different carbon atoms. The carbon atom does not have two OH groups. This differs from (simple) alcohols in which only one hydrogen atom in the hydrocarbon is replaced by an OH group. Polyhydric alcohols with two OH groups are called alkanediols, and polyhydric alcohols with three OH groups are called alkanetriols. Polyhydric alcohols therefore correspond to the general formula [KWj(OH)X, where KW represents a linear or branched, saturated or unsaturated, substituted or unsubstituted hydrocarbon. The substitution can be carried out, for example, by -SH or -NH groups. Preferably, kW is a linear or branched, saturated or unsaturated, unsubstituted hydrocarbon. KW contains at least 2 carbon atoms. Polyhydric alcohols contain 2, 3 or more OH groups (x=2, 3, 4), with only one OH group bonded to each C atom of KW. KW particularly preferably contains 2 to 10, i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Polyhydric alcohols with x=2, 3 or 4 (e.g. pentaerythritol with x=4) can in particular be used. Preference is given to x=2 (alkanediols) and / or x=3 (alkanetriols).

[0054] Particularly preferably, at least one gel phase comprises at least one alkanetriol and / or at least one alkanediol as polyhydric alcohol, in particular at least one C3- to C10-alkanetriol and / or at least one C3- to C10-alkanediol, preferably at least one C3- to Cs-alkanetriol and / or at least one C3- to Cs-alkanediol, in particular at least one C3- to C6-alkanetriol and / or at least one C3- to C5-alkanediol. Preferably, at least one polyhydric alcohol comprises alkanetriol and alkanediol. Thus, in a preferred embodiment, at least one gel phase comprises at least one polymer, in particular PVOH or PVOH with gelatin, and at least one alkanediol and at least one alkanetriol, in particular alkanetriol and alkanediol. Likewise preferred are gel phases which contain at least one polymer, PVOH or PVOH with gelatin, and also C3- to Cs-alkanediols and C3- to Cs-alkanetriols.Furthermore preferred are gel phases which contain at least one polymer, in particular PVOH or PVOH with gelatin, and also C3- to Cs-alkanediols and C3- to C6-alkanetriols.According to the invention, polyhydric alcohols do not include any of their derivatives, such as ethers, esters, etc.

[0055] It has been found that particularly short setting times can be achieved when the corresponding triols (alkanetriols) are combined with the corresponding diols (alkanediols). Furthermore, the gel phase obtained is transparent and has a glossy surface which provides an attractive visual impression.

[0056] Preferably, the automatic dishwashing composition comprises at least one organic solvent in the gel-like phase in an amount of 30 to 90% by weight, in particular 40 to 85% by weight, particularly preferably 50 to 80% by weight, based on the total weight of the gel-like phase.

[0057] The amount of polyhydric alcohol or polyhydric alcohols used in the gel phase according to the invention is preferably at least 45% by weight, in particular 55% by weight or more, with preferred amounts ranging from 45% to 85% by weight, in particular 50% to 80% by weight, based on the total weight of the gel phase.

[0058] Preference is given to the C3-C6-alkanetriols glycerol and / or 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (also called 1,1,1-trimethylolpropane) and / or 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, trishydroxymethylaminoethane) and / or 1,3,5-pentanetriol. Particular preference is given to the C.sub.3-C.sub.6-alkanetriols glycerol and / or 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (also called 1,1-trimethylolpropane). The C.sub.3- C.sub.5-alkanediols are, for example, 1,5-pentanediol, 3-methyl-1,5-pentanediol, 1,4-butanediol, 1,3-propanediol and / or 1,2-propanediol, preferably 1,3-propanediol and / or 1,2-propanediol. Surprisingly, it has been found that the chain length of the diol, in particular the position of the OH groups, influences the transparency of the gel phase. Thus, the OH groups of the diol are preferably not located on directly adjacent carbon atoms. In particular, there are 3 or 4, particularly preferably 3, carbon atoms between two OH groups of the diol.

[0059] The diol 1,3-propanediol is particularly preferred. Surprisingly, it has been found that particularly good results are achieved with mixtures comprising glycerol and 1,3-propanediol and / or 1,2-propanediol, in particular glycerol and 1,3-propanediol.

[0060] According to the invention, a polyethylene glycol having an average molar mass of 200 to 600 g / mol is used in at least one gel phase or in several gel phases, preferably additionally a polyethylene glycol having an average molar mass of about 200 to about 600 g / mol, preferably 300 to 500 g / mol, particularly preferably 350 to 450 g / mol, for example about 400 g / mol (INCI:PEG400), in combination with polyvinyl alcohol.

[0061] Suitable polyethylene glycols have an average molar mass of 300 to 500 g / mol, in particular 350 to 450 g / mol.

[0062] Furthermore, at least one gel phase preferably comprises further anionic polymers, in particular polycarboxylates. These can be used as builder and / or thickening polymers. According to the invention, at least one gel phase may further comprise an anionic polymer or copolymer with builder properties. This is preferably a polycarboxylate. The polycarboxylate used is preferably a copolymer polyacrylate, preferably a sulphopolymer, preferably a copolymer polysulfonate, preferably a hydrophobically modified copolymer polysulfonate. The copolymer may have two, three, four or more different monomer units. Preferred copolymer polysulfonates contain at least one monomer from the group of unsaturated carboxylic acids in addition to the monomer containing sulfonic acid groups.

[0063] Proteases In one embodiment, the disclosure provides one or more subtilisin variants comprising one or more amino acid substitutions as described in more detail below. In some embodiments, the variants provided herein exhibit one or more improved properties, such as improved cleaning performance, or improved stability, or both improved cleaning performance and improved stability, when compared to a subtilisin having the amino acid sequence of SEQ ID NO: 1 or 2. The subtilisin variants provided herein are used in the preparation of cleaning compositions (e.g., automatic dishwashing compositions). In addition, the subtilisin variants provided herein also find use in cleaning methods (e.g., dishwashing methods) that employ such variants or compositions comprising such subtilisin variants.

[0064] Unless otherwise specified herein, one or more subtilisin variants described herein can be made and used by a variety of techniques used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA fields, and industrial enzyme use and development. Terms and abbreviations not defined should be given their ordinary meanings used in the art. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any definitions provided herein should be interpreted in the context of the present specification as a whole. As used herein, the singular forms "a", "an", and "the" include the plural forms unless the context clearly indicates otherwise. Unless otherwise specified, nucleic acid sequences are written left to right in a 5' to 3' orientation and amino acid sequences are written left to right in an amino to carboxy orientation. As used herein, each numerical range includes every narrower numerical range contained within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0065] The term "about" when used herein in connection with a numerical value refers to a range of + / - 0.5 of the numerical value, unless the term is specifically defined in the context. For example, the phrase "a pH value of about 6" refers to a pH value of 5.5 to 6.5, unless the pH value is specifically defined.

[0066] The nomenclature of the amino acid substitutions of one or more of the subtilisin variants described herein uses one or more of the following: position; position: amino acid substitution; or starting amino acid: position: amino acid substitution. Reference to a "position" (e.g., position 5, 8, 17, 22, etc.) encompasses any starting amino acid that may be present at such position, and any substitution that may be present at such position. Reference to a "position: amino acid substitution" (e.g., 1S / T / G, 3G, 17T, etc.) encompasses any starting amino acid that may be present at such position, and one or more amino acids that such starting amino acid may be substituted for. Reference to positions may be recited in several forms, for example, position 003 may also be referred to as position 03 or 3. Reference to a starting amino acid or a substituting amino acid may further be represented as several starting amino acids or substituting amino acids separated by a forward slash (" / "). For example, D275S / K indicates a substitution at position 275 with serine (S) or lysine (K), and P / S197K indicates that the start amino acid proline (P) or serine (S) at position 197 is substituted with lysine (K). Reference to X as an amino acid at a position refers to any amino acid at the recited position.

[0067] The positions of amino acid residues in a given amino acid sequence are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1. That is, the amino acid sequence of SEQ ID NO: 1 serves as a reference sequence for numbering the positions of amino acid residues. For example, the amino acid sequences of one or more subtilisin variants described herein are aligned with the amino acid sequence of SEQ ID NO: 1 using an alignment algorithm described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with the amino acid residues of SEQ ID NO: 1 is conveniently numbered by reference to the number position of its corresponding amino acid residue. For example, a sequence alignment algorithm as described herein identifies one or more positions at which insertions or deletions occur in the subject sequence when compared to a query sequence (sometimes referred to as a "reference sequence"). Sequence alignment with other subtilisin amino acid sequences can be determined, for example, using the amino acid alignment as provided in Figure 1 of International Application No. PCT / US 2017 / 062768, filed November 28, 2018, which claims priority to U.S. Provisional Application No. 62 / 591,976, filed November 29, 2018, entitled "Highly Stable Subtilisin Enzymes."

[0068] The terms "protease" and "proteinase" refer to enzymes capable of degrading proteins and peptides. Proteases have the ability to perform "proteolysis" by hydrolysis of peptide bonds that link together amino acids in the peptide or polypeptide chain that forms the protein. This activity of proteases as protein-digesting enzymes is called "proteolytic activity". There are many well-known procedures for measuring proteolytic activity. For example, proteolytic activity can be confirmed by comparative assays that analyze the ability of each protease to hydrolyze a suitable substrate. Exemplary substrates useful in the analysis of protease or proteolytic activity include, but are not limited to, dimethylcasein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and Keratin Azure (Sigma-Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (see, for example, WO 99 / 34011 and U.S. Pat. No. 6,376,450). The pNA peptidyl assay (see, e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) is also used to determine active enzyme concentration. This assay measures the rate at which p-nitroaniline is released when the enzyme hydrolyzes a soluble synthetic substrate, such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured spectrophotometrically at 405 or 410 nm and is proportional to the active enzyme concentration. Additionally, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a sample of purified protein. Dividing the activity on the substrate by the protein concentration gives the enzyme specific activity.

[0069] As used herein, the genus "Bacillus" includes all species within the genus "Bacillus" known to those of skill in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomic reorganization. Thus, the genus is intended to include reclassified species, including, but not limited to, organisms such as B. stearothermophilus, now called Geobacillus stearothermophilus, or B. polymyxa, now called Paenibacillus polymyxa. The production of resistant endospores under stressful environmental conditions is considered to be the defining characteristic of the genus Bacillus, but this characteristic also applies to the recently named species Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0070] "B. gibsonii subtilisin" includes any subtilisin obtained or derived from a B. gibsonii source. In one embodiment, the subtilisin variants provided herein can be derived from a B. gibsonii-clade subtilisin, such as those described in WO 2015 / 089447, and those described in WO 2016 / 205755. Other B. gibsonii subtilisins include those described in U.S. Patent Application Publication No. 20090275493 and variants thereof, WO 2016 / 087403 and variants thereof, and U.S. Patent No. 7,449,187 and variants thereof. In other embodiments, B. gibsonii subtilisin includes a polypeptide having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 or 2.

[0071] The term "vector" refers to a nucleic acid construct used to introduce or transfer a nucleic acid into a target cell or tissue. A vector is typically used to introduce foreign DNA into a cell or tissue. Vectors include plasmids, cloning vectors, bacteriophages, viruses (e.g., viral vectors), cosmids, expression vectors, shuttle vectors, and the like. A vector typically includes an origin of replication, a multiple cloning site, and a selection marker. The process of inserting a vector into a target cell is typically referred to as transformation. The present invention, in some embodiments, includes a vector comprising a DNA sequence encoding a serine protease polypeptide (e.g., a precursor or mature serine protease polypeptide) operably linked to a suitable prosequence (e.g., secretion, signal peptide sequence, etc.) that can effect expression of the DNA sequence in a suitable host, as well as folding and translocation of the recombinant polypeptide chain.

[0072] As used herein in the context of introducing a nucleic acid sequence into a cell, the term "introduced" refers to any method suitable for transferring a nucleic acid sequence into a cell. Such introduction methods include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic modification of a cell resulting from the uptake, optional genomic integration, and expression of genetic material (e.g., DNA).

[0073] The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid molecule of the present disclosure. Expression may also refer to the translation of mRNA into a polypeptide. Thus, the term "expression" includes any step involved in the "production of a polypeptide", including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0074] The phrase "expression cassette" or "expression vector" refers to a nucleic acid construct or vector, recombinantly or synthetically produced, for the expression of a nucleic acid of interest (e.g., a foreign nucleic acid or a transgene) in a target cell. The nucleic acid of interest typically expresses a protein of interest. An expression vector or expression cassette typically includes a promoter nucleotide sequence that drives or promotes the expression of the foreign nucleic acid. An expression vector or cassette also typically includes other specific nucleic acid elements that allow transcription of the specific nucleic acid in the target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Some expression vectors have the ability to integrate and express heterologous DNA fragments into a host cell or the genome of a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of an appropriate expression vector for the expression of a protein from a nucleic acid sequence incorporated into the expression vector is within the knowledge of one of ordinary skill in the art.

[0075] As used herein, a nucleic acid is "operably linked" to another nucleic acid sequence when it is placed into a functional relationship with the other nucleic acid sequence. For example, a promoter or enhancer is operably linked to a nucleotide coding sequence if the promoter affects the transcription of the coding sequence. A ribosome binding site may be operably linked to a coding sequence if it is positioned so as to facilitate translation of the coding sequence. Typically, "operably linked" DNA sequences are contiguous. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.

[0076] The term "gene" refers to a polynucleotide (e.g., a DNA segment) that encodes a polypeptide and includes regions preceding and following the coding region. In some instances, a gene includes intervening sequences (introns) between individual coding segments (exons).

[0077] The term "recombinant", when used in reference to a cell, typically indicates that the cell has been modified by the introduction of a foreign nucleic acid sequence or that the cell is derived from a cell so modified. For example, a recombinant cell may contain a gene that is not found in the same form within the native (non-recombinant) form of the cell, or a recombinant cell may contain a native gene (found in the native form of the cell) that has been modified and reintroduced into the cell. A recombinant cell may contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell. Such modifications include modifications obtained by gene replacement, site-specific mutagenesis, and related techniques known to those of skill in the art. Recombinant DNA technology includes techniques for the production of recombinant DNA in vitro and techniques by which recombinant DNA may be introduced into cells where it may be expressed or propagated, thereby producing a recombinant polypeptide. "Recombination" and "recombining" of polynucleotides or nucleic acids generally refer to the assembly or combination of two or more nucleic acids or polynucleotide strands or fragments to produce a new polynucleotide or nucleic acid.

[0078] A nucleic acid or polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods known to those of skill in the art, it can be transcribed and / or translated to produce a polypeptide or a fragment thereof. The antisense strand of such a nucleic acid is also said to encode a sequence.

[0079] The terms "host strain" and "host cell" refer to a suitable host for an expression vector containing a DNA sequence of interest.

[0080] A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. The one-letter and three-letter codes for amino acids as defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure. The single letter X refers to any of the 20 amino acids. It is also understood that a polypeptide can be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0081] The term "prosequence" or "propeptide sequence" refers to an amino acid sequence between the signal peptide sequence and the mature protease sequence that is necessary for proper folding and secretion of the protease. They are sometimes called intramolecular chaperones. Cleavage of the prosequence or propeptide sequence yields the mature active protease. Bacterial serine proteases are often expressed as proenzymes. Examples of modified propeptides are provided, for example, in WO 2016 / 205710.

[0082] The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that may be involved in the secretion or direct transport of a mature or precursor form of a protein. A signal sequence is typically located at the N-terminus of a precursor or mature protein sequence. A signal sequence may be endogenous or exogenous. A signal sequence is usually not present in a mature protein. A signal sequence is typically cleaved from a protein by a signal peptidase after the protein has been transported.

[0083] The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide without the signal peptide and propeptide sequences.

[0084] The term "precursor" form of a protein or peptide refers to the mature form of the protein having a prosequence operably linked to the amino- or carbonyl-terminus of the protein. A precursor may also have a "signal" sequence operably linked to the amino-terminus of the prosequence. A precursor may also have additional polypeptides involved in post-translational activity (e.g., polypeptides that are cleaved from the precursor to leave the mature form of the protein or peptide).

[0085] With respect to a polypeptide, the term "wild type" refers to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. Similarly, with respect to a polynucleotide, the term "wild type" refers to a naturally occurring polynucleotide that does not contain an artificial substitution, insertion, or deletion at one or more nucleotides. However, a polynucleotide that encodes a wild type polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes a wild type or parent polypeptide.

[0086] With respect to a polypeptide, the term "parent" includes reference to a naturally occurring or wild-type polypeptide, or a naturally occurring polypeptide in which an artificial substitution, insertion, or deletion has been made at one or more amino acid positions. With respect to a polypeptide, the term "parent" also includes any polypeptide having protease activity that serves as a starting polypeptide for modification, such as substitution, addition, and / or deletion, to result in a variant having one or more modifications compared to the starting polypeptide. That is, a parent polypeptide, or reference polypeptide, is not limited to naturally occurring wild-type polypeptides, but includes any wild-type polypeptide, parent polypeptide, or reference polypeptide. Similarly, with respect to a polynucleotide, the term "parent" can refer to a naturally occurring polynucleotide, or a polynucleotide that includes an artificial substitution, insertion, or deletion at one or more nucleotides. With respect to a polynucleotide, the term "parent" also includes any polynucleotide encoding a polypeptide having protease activity that serves as a starting polynucleotide for modification to result in a variant protease having modifications, such as substitution, addition, and / or deletion, compared to the starting polynucleotide. That is, a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide encoding a wild-type, parent, or reference polypeptide. In some embodiments, a parent polypeptide comprises a B. gibsonii subtilisin. In some embodiments, a parent polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:1.

[0087] The term "naturally occurring" refers, for example, to sequences and residues contained therein that are found in nature (e.g., a polypeptide sequence and the amino acids or nucleotide sequences contained therein and the nucleotides contained therein). Conversely, the term "non-naturally occurring" includes, for example, sequences and residues contained therein that are not found in nature (e.g., a polypeptide sequence and the amino acids or nucleotide sequences contained therein and the nucleic acids contained therein).

[0088] As used herein with reference to an amino acid residue position, "corresponding to" or "corresponds to" or "corresponds" refers to the amino acid residue at the recited position in a protein or peptide, or amino acid residues that are similar, homologous, or equivalent to the recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related or reference protein.

[0089] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by the strain of organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in host organisms containing such DNA sequences. Additionally, the term refers to proteins encoded by DNA sequences of synthetic and / or cDNA origin and having the identifying characteristics of the protein in question. By way of example, "Bacillus-derived protease" refers to an enzyme with proteolytic activity naturally produced by Bacillus, as well as a serine protease produced by a Bacillus source, but produced by other host cells transformed with a nucleic acid encoding the serine protease through the use of genetic engineering techniques.

[0090] The term "identical" in the context of two polynucleotide or polypeptide sequences refers to those nucleotides or amino acids in the two sequences that are the same when aligned for maximum correspondence, as determined using sequence comparison or analysis algorithms described below and known in the art.

[0091] The phrases "% identity" or "percent identity" or "PID" refer to protein sequence identity. Percent identity can be determined using standard techniques known in the art. Percent amino acid identity shared by sequences of interest can be determined by aligning sequences to directly compare sequence information, for example, by using programs such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described, for example, in Altschul et al., J Mol Biol, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993). Percent (%) amino acid sequence identity values ​​are determined by dividing the number of matching identical residues by the total number of residues in the "reference" sequence, including any gaps created by the program for optimal / maximal alignment. The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0092] As used herein, "homologous protein" or "homologous protease" refers to proteins that have clear similarities in primary, secondary, and / or tertiary structure. Protein homology can refer to similarity in linear amino acid sequence when proteins are aligned. Homology can be determined by amino acid sequence alignment, for example, using programs such as BLAST, MUSCLE, or CLUSTAL. Homology searches of protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST with a threshold of 0.001 (E-value cutoff). (Altschul et al., "Gapped BLAST and PSI BLAST a new generation of protein database search programs", Nucleic Acids Res, Set 1;25(17):3389-402(1997)). The BLAST programs use several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences in terms of biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997 and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: adjacent word threshold=11; E-value cutoff=10; scoring matrix=NUC.3.1 (match=1, mismatch=-3); gap opening=5; and gap extension=2. Exemplary default BLAST parameters for amino acid sequence searches include: word size=3; E-value cutoff=10; scoring matrix=BLOSUM62; gap opening=11; and gap extension=1. This information can be used to group protein sequences and / or build phylogenetic trees therefrom.Amino acid sequences can be input into programs such as the Vector NTI Advance suite, and a guide tree can be created using the neighbor-joining (NJ) method (Saitou and Nei, Mol Biol Evol, 4:406-425, 1987). Tree construction can be calculated using Kimura's correction for sequence distances and ignoring positions with gaps. Programs such as AlignX can display the calculated distance values ​​in parentheses following the molecule names displayed on the phylogenetic tree.

[0093] Understanding the homology between molecules can reveal information about the evolutionary history of the molecules as well as their functions. If a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the biochemical function of the new protein. Two molecules are said to be homologous if they are derived from a common ancestor. Homologous molecules, or homologs, can be divided into two classes, paralogs and orthologs. Paralogs are homologs that exist within one species. Paralogs often differ in their detailed biochemical function. Orthologs are homologs that exist in different species and have very similar or identical functions. Protein superfamilies are the largest groupings (clades) of proteins for which a common ancestor can be inferred. Usually, this common ancestor is based on sequence alignment and mechanistic similarity. A superfamily typically contains several protein families, which show sequence similarity within the family. The term "protein clan" is commonly used for protease superfamilies based on the MEROPS protease classification system. As used herein, the term "subtilisin" includes any member of the S8 serine protease family described in MEROPS-The Peptidase Database (Rawlings, ND, et al (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 44, D343-D350).

[0094] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994). The default parameters of the CLUSTAL W algorithm include: gap opening penalty = 10.0; gap extension penalty = 0.05; protein weight matrix = BLOSUM series; DNA weight matrix = IUB; delayed divergent sequence % = 40; gap separation distance = 8; DNA transition weight = 0.50; list of hydrophilic residues = GPSNDQEKR; use negative matrix = OFF; residue specific penalty = switch ON; hydrophilicity penalty = switch ON; and terminal gap separation penalty = switch OFF. The CLUSTAL algorithm includes deletions that occur at either end. For example, a variant with a 5 amino acid deletion at either end (or within the polypeptide) of a 500 amino acid polypeptide will have a percent sequence identity of 99% (495 / 500 identical residues x 100) to the "reference" polypeptide. Such variants are encompassed by variants having "at least 99% sequence identity" to the polypeptide.

[0095] A nucleic acid or polynucleotide is "isolated" when it is at least partially or completely separated from other components, including, for example, but not limited to, other proteins, nucleic acids, cells, etc. Similarly, a polypeptide, protein, or peptide is "isolated" when it is at least partially or completely separated from other components, including, for example, but not limited to, other proteins, nucleic acids, cells, etc. On a molar basis, an isolated species is more abundant than other species in a composition. For example, an isolated species may comprise at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% (on a molar basis) of all macromolecular species present. Preferably, the species of interest is purified to essential homogeneity (i.e., contaminating species cannot be detected in the composition by conventional detection methods). Purity and homogeneity can be determined using a number of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of the nucleic acid or protein sample, respectively, followed by visualization by staining. If desired, high resolution techniques, such as high performance liquid chromatography (HPLC) or similar means, can be utilized to purify the material.

[0096] The term "purified" as applied to a nucleic acid or polypeptide generally refers to a nucleic acid or polypeptide that is essentially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a distinct band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule if the concentration of the molecule is significantly increased following application of a purification or concentration technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other particular substance or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0097] The term "cleaning activity" refers to the cleaning performance achieved by a serine protease polypeptide, variant, or reference subtilisin under conditions prevailing during proteolytic, hydrolytic, cleaning, or other processes of the present disclosure. In some embodiments, the cleaning performance of a serine protease or reference subtilisin may be determined by using various assays for cleaning one or more enzyme-sensitive stains (e.g., stains due to food, grass, blood, ink, milk, oil, and / or egg proteins) on an article or surface. The cleaning performance of one or more subtilisin variants or reference subtilisins described herein may be determined by subjecting a stain on an article or surface to standard cleaning conditions and evaluating the extent to which the stain is removed by using various chromatographic, spectrophotometric, or other quantitative methodologies. Exemplary cleaning assays and methods are known in the art and include, but are not limited to, those described in WO 99 / 34011 and U.S. Pat. No. 6,605,458, as well as the cleaning assays and methods included in the Examples provided below.

[0098] The term "effective amount" of one or more subtilisin variants or reference subtilisins described herein refers to an amount of protease that achieves a desired level of enzymatic activity in a particular cleaning composition. Such effective amounts are readily ascertainable by one of skill in the art and are based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granule, tablet, bar) composition is desired.

[0099] Disclosed herein are one or more subtilisin variants useful in cleaning applications and methods, as well as in various industrial applications. Also disclosed herein are one or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin variants. In some embodiments, one or more subtilisin variants described herein can be incorporated into cleaning compositions useful in cleaning applications and methods for cleaning articles or surfaces in need of cleaning.

[0100] The protease has the following sequence with respect to SEQ ID NO:1: (i) a mutation at position 122, (ii) one or more mutations at positions 126, 127, 128, 211, and 212; A subtilisin variant comprising: The variant has at least 80% identity with the amino acid sequence of SEQ ID NO:1.

[0101] Preferably, the mutant is (i) a mutation at position M122L, (ii) one or more mutations at positions 126, 127, 128, 211, and 212; Includes.

[0102] Preferably, the variant comprises two or more mutations at positions 126, 127, 128, 211 and 212, preferably three or more mutations at positions 126, 127, 128, 211 and 212, preferably four or more mutations at positions 126, 127, 128, 211 and 212, preferably five mutations at positions 126, 127, 128, 211 and 212.

[0103] Preferably the mutant comprises one or more mutations selected from X126A, X127E, X128G, X211Q and X212Q, preferably two or more mutations selected from X126A, X127E, X128G, X211Q and X212Q, preferably three or more mutations selected from X126A, X127E, X128G, X211Q and X212Q, preferably four or more mutations selected from X126A, X127E, X128G, X211Q and X212Q, preferably five mutations selected from X126A, X127E, X128G, X211Q and X212Q.

[0104] Preferably, the variants include the following mutations: X122L, X126A, X127E, X128G, X211Q and X212Q.

[0105] Preferably the mutant comprises one or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably two or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably three or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably four or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably five mutations selected from S126A, D127E, F128G, M211Q and N212Q.

[0106] Preferably, the mutant comprises the mutations M122L, S126A, D127E, F128G, M211Q and N212Q.

[0107] In another embodiment a subtilisin variant is provided, wherein the variant comprises i) at least one, two, three, four or five substitutions selected from the group consisting of S039E, S099R, S126A, D127E and F128G, and ii) one or more additional substitutions selected from the group consisting of N74D, T114L, M122L, M122I, N198A, M211Q, N212Q and N242D, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0108] In another embodiment, a subtilisin variant is provided, wherein the variant comprises amino acid substitutions X039E-X099R-X126A-X127E-X128G and further comprises one or more additional substitutions at one, two, three or more positions selected from the group consisting of 74, 114, 122, 198, 211, 212 and 242, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1. In some embodiments herein, references to substitutions X039E, X099R, X126A, X127E and X128G include S039E, S099R, S126A, D127E and F128G.

[0109] In another embodiment, a subtilisin variant is provided, wherein the variant comprises an amino acid substitution selected from one or more substitutions selected from X039E, X099R, X126A, X127E, and X128G, and further comprises one or more additional substitutions selected from the group consisting of X74D, X114L, X122L, X122I, X198A, X211Q, X212Q, and X242D, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1.

[0110] In another embodiment, a subtilisin variant is provided, wherein the variant comprises an amino acid substitution selected from one or more substitutions selected from S039E, S099R, S126A, D127E, and F128G, and further comprises one or more additional substitutions selected from the group consisting of N74D, T114L, M122L, M122I, N198A, M211Q, N212Q, and N242D, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1.

[0111] In another embodiment a subtilisin variant is provided, wherein the variant comprises i) two or more amino acid substitutions selected from the group consisting of S039E, N74D, S099R, N242D, and ii) one or more additional substitutions selected from the group consisting of T114L, M122L, M122I, S126A, F128G, N198A, M211Q, N212Q, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1.

[0112] In another embodiment, the variant is S039E-S099R-S126A-D127E-F128G-M211Q-N242D, S039E-N074D-S099R-M122L-S126A-D127E-F128G-N198A-M211Q-N212Q, S039E-N074D-S099R-M122L-S126A-D127E-F128G-N198A-M211Q-N212Q-N242D, S039E-N074D-S099R-S126A-D127E-F128G-M211Q-N212Q-N242D, S039E-N074D -S099R-S126A-D127E-F128G-N198A-M211Q-N212Q-N242D, S039E-N074D-S 099R-S126A-D127E-F128G-N198G, S039E-N074D-S099R-T114L-S126A-D12 7E-F128G, S039E-N074D-S099R-T114L-M122L-S126A-D127E-F128G-N198A -M211Q-N212Q, S039E-N074D-S099R-T114L-M122L-S126A-D127E-F128G-N 198A-M211Q-N212Q-N242D, S039E-N074D-S099R-T114L-S126A-D127E-F12 8G-M211E, S039E-N074D-S099R-T114L-S126A-D127E-F128G-M211E-N242D , S039E-N074D-S099R-T114L-S126A-D127E-F128G, M211Q, S039E-N074D-S 099R-T114L-S126A-D127E-F128G-M211Q-N212Q-N242D, S039E-N074D-S09 9R-T114L-S126A-D127E-F128G-N198A-M211Q-N212Q, S039E-N074D-S099R -T114L-S126A-D127E-F128G-N198A-M211Q-N212Q-N242D, S039E-S099R-S 126A-D127E-F128G-N198G-M211Q-N212Q, S039E-S099R-T114L-S126A-D12 7E-F128G-M211E, S039E-S099R-T114L-S126A-D127E-F128G-M211E-N212Q,S039E-S099R-T114L-S126A-D127E-F128G-M211E-N242D, S039E-S099R-T114L-S126A-D127 E-F128G-M211Q, S039E-S099R-T114L-S126A-D127E-F128G-M211Q-N212Q-N242D, S039E-S0 Subtilisin variants are provided that contain a set of substitutions selected from the group consisting of 99R-T114L-S126A-D127E-F128G-M211Q-N242D, and S039E-S099R-T114L-S126A-D127E-F128G-N242D, wherein the amino acid positions are numbered according to the amino acid sequence of SEQ ID NO:1.

[0113] Another embodiment relates to one or more subtilisin variants described herein, with the proviso that one or more of the substitutions are not naturally occurring. Yet a further embodiment relates to one or more subtilisin variants described herein, wherein said variant (i) is B. gibsonii BG46 subtilisin, (ii) is isolated, (iii) has proteolytic activity, or (iv) comprises a combination of (i)-(iii). A further embodiment relates to one or more subtilisin variants as described herein, said variants being derived from a parent or reference polypeptide having (i) 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2; or (ii) 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2. In yet another embodiment, the parent comprises the amino acid sequence of SEQ ID NO: 1 or 2. A further embodiment relates to one or more subtilisin variants as described herein, said variants being derived from a parent or reference polypeptide having (i) 60%, 65%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2. (ii) less than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1; (iii) less than 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. The present invention relates to a subtilisin variant comprising an amino acid sequence having the following structure:

[0114] In some embodiments, the subtilisin parent or variant molecule provided herein may also comprise one or more of the following: X012E / L / V, X021V, X025R, X037E, X039T, X041F, X043V, X044P, X060D, X078D, X079L, X084A, X087E, X097D, X099E, X101G, X012L, X107E, X115D, X117I, X118N, X122L, X127P, X142G, X145S, and at least one, two, three or more additional substitutions selected from X149S, X154D, X156A, X160S, X167D, X174A, X175N, X176E, X177E / I / V, X185E, X188A, X200E, X205D, X208N, X209N, X211L / N / S, X212D / H / N, X222S, X228I, X230E / H, X236D, X247N, X250D, and X253D / P. Examples of such one, two, three or more substitution combinations that can be combined with the variants provided herein include X253D-X256E, X025R-X117I-X118N, X044P-X175N-X208N-X230H, X041F-X078D-X084A, X101G-X174A, X021V-X177I, X021V-X142G-X188A, X021V-X122L-X222S, X012L-X021V-X122L-X222S, X0 21V-X122L-X253D, X021V-X177V-X228I, X021V-X039T-X122L-X177E, X021V-X079L-X087E -X209N-X222S, X021V-X122L-X222S-X247N, X021V-X122L, X039E-X074D-X087E, X039E-X 074D-X087E-X253D, X021V-X039E-X074D-X087E-X253D, X039E-X074D-X087E-X122L-X25 3D, X021V-X039E-X074D-X087E-X122L-X253D, X097D-X099E, X122L-X145S-X156A, X211N-X212D, X211L-X212D, X127P-X211L-X212D, and X012L-X122L-X222S.

[0115] The present disclosure includes subtilisin variants having one or more modifications to surface exposed amino acids. The surface modifications in the enzyme variants may be useful in detergent compositions, with at least one of these properties being improved from the parent subtilisin enzyme, with a minimum figure of merit for cleaning performance, enzyme stability in detergent compositions, and enzyme thermostability. In some embodiments, the surface modification changes the hydrophobicity and / or charge of the amino acid at that position. Hydrophobicity can be determined using techniques known in the art, such as those described in White and Wimley (White, SH and Wimley, WC,. (1999) Annu. Rev. Biophys. Biomol. Struct. 28:319-65). As used herein, "surface properties" can be used in reference to properties such as electrostatic charge, and hydrophobicity and hydrophilicity exhibited by protein surfaces.

[0116] In still further embodiments, one or more of the subtilisin variants described herein have one or more improved properties when compared to a reference or parent subtilisin, the improved properties being selected from improved cleaning performance, improved stability in detergents, and combinations thereof.

[0117] In the context of oxidative, chelating, denaturing, detergent, thermostable and / or pH-stable proteases, the term "enhanced stability" or "improved stability" refers to a higher degree of retained proteolytic activity over time compared to a reference protease, e.g., a wild-type or parent protease. Autolysis has been identified as one mode of subtilisin activity loss in liquid detergents. Stoner et al., 2004 Protease autolysis in heavy-duty liquid detergent formulations: effects of thermodynamic stabilizers and protease inhibitors, Enzyme and Microbial Technology 34:114-125).

[0118] With respect to protease variants, the terms "thermostable" and "thermostable" and "thermostable" refer to a protease that retains a greater amount of residual activity when compared to a parent or reference protease after exposure to altered temperatures for a given period of time under conditions prevailing during proteolysis, hydrolysis, cleaning or other processes (or "stress conditions"). Residual activity is the amount of activity remaining after testing compared to the initial activity of the sample and can be reported as a percentage, e.g., % residual activity. "Altered temperature" encompasses elevated or decreased temperatures. In some embodiments, the protease variants provided herein are incubated at a temperature between 40° C. and 80° C. for a period of time, e.g., at least about 5 minutes, at least about 20 minutes, at least about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, about 660 minutes, about 720 minutes, about 780 minutes, about retains at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% proteolytic activity after exposure for 840 minutes, about 900 minutes, about 960 minutes, about 1020 minutes, about 1080 minutes, about 1140 minutes, or about 1200 minutes.

[0119] The subtilisin variants provided herein can be used in the production of various compositions, such as enzyme compositions and cleaning or detergent compositions. The enzyme compositions include the subtilisin variants provided herein. The enzyme compositions can be in any form, such as granules, liquid formulations, or enzyme slurries.

[0120] Enzyme granules can be made, for example, by rotary atomization, wet granulation, dry granulation, spray drying, disk granulation, extrusion, pan coating, spheronization, drum granulation, fluidized bed agglomeration, high shear granulation, fluidized bed spray coating, crystallization, precipitation, emulsion gelation, rotary disk atomization and other casting approaches, as well as the prilling process. The core of the granule can be the granule itself or the inner core of a layered granule.

[0121] The core may include one or more water-soluble or dispersible agents, including, but not limited to, sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives, such as hydroxyl-propyl-methylcellulose, carboxy-methylcellulose, and hydroxyl-ethylcellulose), phosphates, calcium, protease inhibitors, and combinations thereof. Suitable dispersible agents include, but are not limited to, clays, nonpareils (combinations of sugars and starches; e.g., starch-sucrose-nonpareils-ASNP), talc, silicates, carboxymethylcellulose, starch, and combinations thereof.

[0122] In some embodiments, the core comprises primarily sodium sulfate. In some embodiments, the core consists essentially of sodium sulfate. In certain embodiments, the core consists solely of sodium sulfate.

[0123] In some embodiments, the core comprises a subtilisin variant provided herein. In other embodiments, the core comprises one or more enzymes in addition to a protease. In other embodiments, the core is inactive and does not contain an enzyme.

[0124] In some embodiments, the core is an enzyme powder that includes a UFC containing an enzyme. The enzyme powder may be spray dried and may optionally be mixed with any of the water-soluble or dispersible agents listed herein. The enzyme may be or may include a protease that is to be stabilized, in which case the enzyme powder should further include a stabilizer.

[0125] In some embodiments, the core is coated with at least one coating layer. In certain embodiments, the core is coated with at least two coating layers. In another specific embodiment, the core is coated with at least three coating layers. The materials used for the coating layers may be suitable for use in cleaning and / or detergent compositions (see, for example, US20100124586, WO9932595 and US5324649).

[0126] In some embodiments, the coating layer comprises one or more of the following materials: inorganic salts (e.g., sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, sugars (e.g., sucrose, lactose, glucose, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives, such as hydroxyl-propyl-methylcellulose, carboxy-methylcellulose, and hyaluronan). Examples of suitable coating layer additives include hydroxyl-ethylcellulose, clays, nonpareils (combinations of sugars and starches), silicates, carboxymethylcellulose, phosphates, starches (e.g., corn starch), fats, oils (e.g., rapeseed oil, and paraffin oil), lipids, vinyl polymers, vinyl copolymers, polyvinyl alcohol (PVA), plasticizers (e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate, and water), anti-agglomerating agents (e.g., talc, clays, amorphous silica, and titanium dioxide), anti-foaming agents (e.g., FOAMBLAST 882® and EROL 6000K®), and talc. US 20100124586, WO 9932595, and US 5324649 detail suitable ingredients for the coating layer.

[0127] In some embodiments, the coating layer comprises a sugar (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin, and fructose). In some embodiments, the coating layer comprises a polymer, such as polyvinyl alcohol (PVA). PVA suitable for incorporation into the coating layer of the multi-layer granules includes partially hydrolyzed, fully hydrolyzed, and moderately hydrolyzed, and has low to high viscosity. In some embodiments, the coating layer comprises an inorganic salt, such as sodium sulfate.

[0128] In some embodiments, at least one coating layer is an enzyme coating layer. In some embodiments, the core is coated with at least two enzyme layers. In other embodiments, the core is coated with at least three or more enzyme layers.

[0129] In some embodiments, the enzyme is an acyltransferase, amylase, α-amylase, β-amylase, α-galactosidase, arabinase, arabinosidase, arylesterase, β-galactosidase, β-glucanase, carrageenase, catalase, cellulase, chondroitinase, cutinase, dispersin, endoglucanase, endo-β-mannanase, exo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipolytic enzyme, lipoxygenase, lysozyme, mannanase, mesylate, glyceryl esterase ... Subtilisin variants as provided herein in combination with one or more additional enzymes selected from the group consisting of taroproteases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, PETases, phenoloxidases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonase, tannase, transglutaminase, xylan acetyl-esterases, xylanases, and xylosidases; and combinations or mixtures thereof. Generally, at least one enzyme coating layer comprises at least one protease.

[0130] The above list of enzymes is merely exemplary and is not meant to be exhaustive. Any enzyme can be used in the granules described herein, including wild-type, recombinant and mutant enzymes of bacterial, fungal, yeast origin, as well as acidic, neutral or alkaline enzymes.

[0131] Another embodiment relates to a method of cleaning a surface, comprising contacting a surface or an article in need of cleaning with an effective amount of one or more subtilisin variants provided herein, or a composition containing one or more subtilisin variants provided herein. In some embodiments, the surface or article in need of cleaning comprises a proteinaceous stain on the surface. In some embodiments, the surface or article in need of cleaning comprises a proteinaceous or creme brûlée, or BMI, or egg or baked cheese stain. The term "stain" includes any type of soil on the surface of an article, such as a hard surface article (e.g., dishware) or a fabric. In some embodiments, the stain is a proteinaceous stain. As used herein, a "proteinaceous stain" is a stain or soil that contains protein.

[0132] Further embodiments relate to methods of cleaning proteinaceous stains comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein, or a composition containing one or more subtilisin variants provided herein.

[0133] Another embodiment relates to a method of cleaning crème brûlée, comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein, or a composition containing one or more subtilisin variants provided herein.

[0134] Another embodiment relates to a method of cleaning egg or egg yolk stains comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein, or a composition containing one or more such subtilisin variants.

[0135] Another embodiment relates to a method of cleaning baked cheese stains comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein, or a composition containing one or more such subtilisin variants.

[0136] Another embodiment relates to a method of cleaning BMI brulee comprising contacting a surface or item in need of cleaning with an effective amount of one or more subtilisin variants provided herein, or a composition containing one or more such subtilisin variants.

[0137] In still further embodiments, the one or more subtilisin variants used in the methods described herein comprise an amino acid sequence having less than 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2. In yet another embodiment, the one or more subtilisin variants used in the methods for cleaning creme brulee stains described herein have a creme brulee stain cleaning PI > 1.1 when compared to SEQ ID NO: 1 or 2. In yet another embodiment, one or more subtilisin variants used in the methods for cleaning Crème Brulee stains described herein have a Crème Brulee stain cleaning PI > 1.1 when compared to SEQ ID NO: 1 or 2, and the Crème Brulee stain cleaning performance of said variants is measured according to the Crème Brulee assay described in Example 2. Yet another embodiment relates to a method for cleaning Crème Brulee stains described herein, with the proviso that one or more subtilisins used in said method comprise one or more non-naturally occurring substitutions. In yet another embodiment, one or more subtilisin variants used in the methods for cleaning baked cheese stains described herein have a baked cheese stain cleaning PI > 1.1 when compared to SEQ ID NO: 1 or 2. In yet another embodiment, one or more subtilisin variants used in the methods of cleaning baked cheese stains described herein have a baked cheese stain cleaning PI > 1.1 when compared to SEQ ID NO: 1 or 2, and the baked cheese stain cleaning performance of the variants is measured according to the baked cheese assay described in Example 2. Yet another embodiment is a method of cleaning baked cheese stains described herein, with the proviso that one or more subtilisins used in the aforementioned method comprise one or more non-naturally occurring substitutions. In yet another embodiment, one or more subtilisin variants used in the methods of cleaning egg yolk stains described herein have an egg yolk stain cleaning PI > 1.1 when compared to SEQ ID NO: 1 or 2.In yet another embodiment, one or more subtilisin variants used in the methods of cleaning egg yolk stains described herein have an egg yolk stain cleaning PI > 1.1 when compared to SEQ ID NO: 1 or 2, and the egg yolk stain cleaning performance of the variants is measured according to the egg yolk assay described in Example 2. Yet another embodiment is a method of cleaning egg yolk stains described herein, wherein one or more subtilisins used in the aforementioned methods comprise one or more non-naturally occurring substitutions. In yet another embodiment, one or more subtilisin variants used in the methods of cleaning BMI stains described herein have a BMI stain cleaning PI > 1.1 when compared to SEQ ID NO: 1 or 2. In yet another embodiment, one or more subtilisin variants used in the methods of cleaning BMI stains described herein have a BMI stain cleaning PI > 1.1 when compared to SEQ ID NO: 1 or 2, and the BMI stain cleaning performance of the variants is measured according to the BMI assay described in Example 2. Yet another embodiment is a method of cleaning BMI stains as described herein, except that one or more subtilisins used in the method comprise one or more non-naturally occurring substitutions. In a further embodiment, one or more subtilisin variants used in the method described herein are (i) isolated, (ii) have proteolytic activity, or (iii) comprise a combination of (i) and (ii).

[0138] In another embodiment, the variants provided herein include one or more variants having an amino acid substitution selected from the group consisting of those listed in Table 5, which have a PI > 1.1 in one or more of cleaning assays, including laundry and dish assays such as BMI, egg, creme brûlée, and / or baked cheese assays, compared to a parent subtilisin having the amino acid sequence of SEQ ID NO: 1 or 2, or have a higher residual activity in an EDTA stability assay than that of the parent subtilisin or a reference subtilisin.

[0139] One or more subtilisin variants described herein may be subjected to various changes, such as insertion, deletion, and / or substitution of one or more amino acids, either conservative or non-conservative, including cases where such changes do not substantially change the enzymatic activity of the variant. Similarly, the nucleic acids of the present invention may also be subjected to various changes, such as one or more substitutions of one or more nucleotides in one or more codons, such that a particular codon encodes the same or different amino acid, resulting in either a silent mutation (e.g., where the encoded amino acid is not altered by the nucleotide mutation) or a non-silent mutation; one or more deletions of one or more nucleotides (or codons) in the sequence; one or more additions or insertions of one or more nucleotides (or codons) in the sequence; and / or one or more truncations or truncations of one or more nucleotides (or codons) in the sequence. Many such changes in the nucleic acid sequence may not substantially change the enzymatic activity of the resulting encoded polypeptide enzyme, as compared to the polypeptide enzyme encoded by the original nucleic acid sequence. The nucleic acid sequences described herein can also be modified to include one or more codons that provide for optimal expression in an expression system (e.g., a bacterial expression system), while said one or more codons still encode the same amino acid, if desired.

[0140] Described herein are one or more isolated, non-naturally occurring, or recombinant polynucleotides comprising a nucleic acid sequence encoding one or more subtilisin variants described herein, or a recombinant polypeptide or active fragment thereof. One or more nucleic acid sequences described herein are useful for recombinant production (e.g., expression) of one or more subtilisin variants described herein, typically by expression of a plasmid expression vector comprising a sequence encoding one or more subtilisin variants described herein, or a fragment thereof. One embodiment provides a nucleic acid encoding one or more subtilisin variants described herein, wherein the variants are mature forms having proteolytic activity. In some embodiments, one or more subtilisin variants described herein are recombinantly expressed with a homologous propeptide sequence. In other embodiments, one or more subtilisin variants described herein are recombinantly expressed with a heterologous propeptide sequence (e.g., B. lentus (SEQ ID NO: 5)).

[0141] One or more of the nucleic acid sequences described herein can be produced by using any suitable synthesis, manipulation, and / or isolation technique, or a combination thereof. For example, one or more of the polynucleotides described herein can be produced using standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques, well known to those skilled in the art. In such techniques, fragments of up to 50 or more nucleotide bases are typically synthesized and then joined (e.g., by enzymatic or chemical ligation methods) to form essentially any desired contiguous nucleic acid sequence. The synthesis of one or more of the polynucleotides described herein can also be facilitated by any suitable method known in the art, including, but not limited to, chemical synthesis using classical phosphoramidite methods (see, e.g., Beaucage et al. Tetrahedron Letters 22:1859-69 (1981)), or the method described in Matthes et al., EMBO J.3:801-805 (1984), as typically implemented in automated synthesis methods. One or more polynucleotides described herein can also be produced by using an automated DNA synthesizer. Customized nucleic acids can be obtained from a variety of commercial sources (e.g., ATUM (DNA 2.0), Newark, CA, USA; Life Tech (GeneArt), Carlsbad, CA, USA; GenScript, Ontario, Canada; Base Clear BV, Leiden, The Netherlands; Integrated DNA Technologies, Skokie, IL, USA; Ginkgo Bioworks (Gen9), Boston, MA, USA and Twist Bioscience, San Francisco, CA, USA). Other techniques for synthesizing nucleic acids and related principles are described, for example, in Itakura et al., Ann. Rev. Biochem. 53:323 (1984) and Itakura et al., Science 198:1056 (1984).

[0142] Recombinant DNA techniques useful for modifying nucleic acids are well known in the art, such as restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more polynucleotides described herein can also be obtained by screening a cDNA library using one or more oligonucleotide probes that can hybridize to or PCR amplify a polynucleotide encoding one or more subtilisin variants described herein, or a recombinant polypeptide or active fragment thereof. Procedures for screening and isolating cDNA clones, as well as PCR amplification procedures, are well known to those skilled in the art and are described in standard references known to those skilled in the art. One or more polynucleotides described herein can be obtained by modifying a naturally occurring polynucleotide backbone (e.g., one encoding one or more subtilisin variants or reference subtilisins described herein), for example, by known mutagenesis procedures (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro recombination). A variety of methods suitable for generating modified polynucleotides described herein that encode one or more subtilisin variants described herein are known in the art, including, but not limited to, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-specific mutagenesis, and directed evolution, as well as a variety of other recombinant approaches.

[0143] Further embodiments include one or more vectors comprising one or more subtilisin variants described herein (e.g., polynucleotides encoding one or more subtilisin variants described herein); expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequences described herein; isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequences described herein; isolated or recombinant cells comprising one or more polynucleotide sequences described herein; and compositions comprising one or more such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof. Regarding.

[0144] Some embodiments relate to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein that contain one or more nucleic acid or polynucleotide sequences described herein. Some such recombinant cells are transformed or transfected with such at least one vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells include bacterial cells, including, but not limited to, cells of Bacillus species, such as B. subtilis cells. Other embodiments relate to recombinant cells (e.g., recombinant host cells) that comprise one or more subtilisins described herein.

[0145] In some embodiments, one or more of the vectors described herein are expression vectors or expression cassettes that include one or more of the polynucleotide sequences described herein operably linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., a promoter operably linked to one or more of the polynucleotide sequences described herein). The vectors may include a transcription terminator and / or a selection gene (e.g., an antibiotic resistance gene) that allows for continuous culture maintenance of plasmid-infected host cells by growth in antimicrobial-containing medium.

[0146] Expression vectors can be derived from plasmid or viral DNA, or in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (Harwood and Cutting, [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990); suitable replicating plasmids for B. subtilis include those listed on p. 92). (See also Perego, "Integrational Vectors for Genetic Manipulations in Bacillus subtilis"; Sonenshein et al., [eds.]; "Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics", American Society for Microbiology, Washington, DC (1993), pp. 615-624); and p2JM103BBI).

[0147] For expression and production of a protein of interest (e.g., one or more subtilisin variants described herein) in a cell, one or more expression vectors containing one or more copies, and in some cases multiple copies, of a polynucleotide encoding one or more subtilisin variants described herein are transformed into the cell under conditions suitable for expression of the variants. In some embodiments, the polynucleotide sequence encoding one or more subtilisin variants described herein (as well as other sequences contained in the vector) is integrated into the genome of the host cell, while in other embodiments, the plasmid vector containing the polynucleotide sequence encoding one or more subtilisin variants described herein remains within the cell as an autonomous extrachromosomal element. Some embodiments provide both an extrachromosomal nucleic acid element and an input nucleotide sequence that is integrated into the host cell genome. The vectors described herein are useful for production of one or more subtilisin variants described herein. In some embodiments, the polynucleotide construct encoding one or more subtilisin variants described herein is present on an integration vector that allows for integration and optionally amplification of the polynucleotide encoding the variant into the host chromosome. Examples of sites for integration are well known to those of skill in the art. In some embodiments, transcription of a polynucleotide encoding one or more of the subtilisin variants described herein is accomplished by a promoter that is the wild-type promoter of the parent subtilisin. In some other embodiments, the promoter is heterologous to one or more of the subtilisin variants described herein, but is functional in the host cell.Exemplary promoters for use in bacterial host cells include the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters; the promoter of the B. stearothermophilus maltogenic amylase gene; the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene; the B. pumilus xylosidase gene; B. thuringiensis cryIIIA; and the B. licheniformis α-amylase gene. Additional promoters include, but are not limited to, the A4 promoter, as well as the phage lambda PR or PL promoters and the E. coli lac, trp or tac promoters.

[0148] One or more subtilisin variants described herein may be produced in any suitable microbial host cell, including bacteria and fungi. In some embodiments, one or more subtilisin variants described herein may be produced in a Gram-positive bacteria. In some embodiments, the host cell is a Bacillus spp., Streptomyces spp., Escherichia spp., Aspergillus spp., Trichoderma spp., Pseudomonas spp., Corynebacterium spp., Saccharomyces spp., or Pichia spp. In some embodiments, one or more subtilisin variants described herein are produced by a Bacillus spp. host cell. Examples of Bacillus species host cells used to produce one or more subtilisin variants described herein include, but are not limited to, B. licheniformis, B. gibsonii, B. lentus, B. subtilis, B. amyloliquefaciens, B. brevis, B. stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B. pumilis, B. thuringiensis, B. clausii, and B. megaterium, as well as other organisms within the Bacillus genus. In some embodiments, B. subtilis host cells are used to produce the variants described herein. U.S. Patent Nos. 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus host strains that can be used to produce one or more subtilisin variants described herein, although other suitable strains can be used.

[0149] Some bacterial strains that can be used to produce one or more subtilisin variants described herein include non-recombinant (i.e., wild-type) Bacillus spp. strains, as well as variants of naturally occurring and / or recombinant strains. In some embodiments, the host strain is a recombinant strain, where a polynucleotide encoding one or more subtilisin variants has been introduced into the host. In some embodiments, the host strain is a B. subtilis host strain, particularly a recombinant B. subtilis host strain. Many B. subtilis strains are known, such as strains 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753-PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211 (see, e.g., Hoch et al., Genetics, 2001). 73:215-228 (1973); see also U.S. Pat. No. 4,450,235; U.S. Pat. No. 4,302,544, and European Patent No. 0134048. The use of B. subtilis as an expression host cell is well known in the art (see, e.g., Palva et al., Gene 19:81-87 (1982); Fahnestock and Fischer, J. Bacteriol., 165:796-804 (1986); and Wang et al., Gene 69:39-47 (1988)).

[0150] In some embodiments, the Bacillus host cell is a Bacillus species that includes a mutation or deletion in at least one of the following genes: degU, degS, degR, and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU(Hy)32 (see, e.g., Msadek et al., J. Bacteriol. 172:824-834 (1990); and Olmos et al., Mol. Gen. Genet. 253:562-567 (1997)). In some embodiments, the Bacillus host comprises a mutation or deletion in scoC4 (see, e.g., Caldwell et al., J. Bacteriol. 183:7329-7340 (2001)); spoIIE (see, e.g., Arigoni et al., Mol. Microbiol. 31:1407-1415 (1999)); and / or oppA or other genes of the opp operon (see, e.g., Perego et al., Mol. Microbiol. 5:173-185 (1991)). Indeed, any mutation in the opp operon that causes the same phenotype as a mutation in the oppA gene is contemplated for use in some embodiments of the altered Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, modified Bacillus host cell strains that can be used to produce one or more subtilisin variants described herein are Bacillus host strains that already contain a mutation in one or more of the above-mentioned genes. Additionally, Bacillus spp. host cells are used that contain mutations and / or deletions of endogenous protease genes. In some embodiments, the Bacillus spp. host cells contain a deletion of the aprE and nprE genes. In other embodiments, the Bacillus spp. host cells contain a deletion of five protease genes, while in other embodiments, the Bacillus spp. host cells contain a deletion of nine protease genes (see, e.g., U.S. Patent Application Publication No. 2005 / 0202535).

[0151] A host cell is transformed with one or more nucleic acid sequences encoding one or more subtilisin variants described herein using any suitable method known in the art. Methods for introducing nucleic acids (e.g., DNA) into Bacillus or E. coli cells using plasmid DNA constructs or vectors, and for transforming such plasmid DNA constructs or vectors into such cells, are well known. In some embodiments, the plasmid is then isolated from the E. coli cell and transformed into the Bacillus cell. However, it is not necessary to use an intermediate microorganism such as E. coli, and in some embodiments, the DNA construct or vector is directly introduced into the Bacillus host.

[0152] Exemplary methods for introducing one or more of the nucleic acid sequences described herein into a Bacillus cell can be found, for example, in Ferrari et al., "Genetics," in Harwood et al. [eds.], Bacillus, Plenum Publishing Corp. (1989), pp. 57-72; Saunders et al., J. Bacteriol. 157:718-726 (1984); Hoch et al., J. Bacteriol. 93:1925-1937 (1967); Mann et al., Current Microbiol. 13:131-135 (1986); Holubova, Folia Microbiol. 30:97 (1985); Chang et al., Mol. Gen. Genet. 168:11-115 (1979); Vorobjeva et al., FEMS Microbiol. Lett. 7:261-263 (1980); Smith et al., Appl. Env. Microbiol. 51:634 (1986); Fisher et al., Arch. Microbiol. 139:213-217 (1981); and McDonald, J. Gen. Microbiol. 130:203 (1984). Indeed, methods such as transformation, including protoplast transformation and transfection, transduction, and protoplast fusion, are well known and suitable for use herein. Methods known in the art for transforming Bacillus cells include methods such as plasmid marker rescue transformation, which involves uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (see Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol. 154:1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169:1205-1211 (1987)).In this method, an input donor plasmid recombines with a homologous region of a resident "helper" plasmid in a process that mimics chromosomal transformation.

[0153] In addition to commonly used methods, in some embodiments, a host cell is directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more subtilisin variants described herein (i.e., no intermediate cells are used to amplify or otherwise carry out the DNA construct or vector prior to introduction into the host cell). Introduction of a DNA construct or vector described herein into a host cell includes physical and chemical methods known in the art for introducing a nucleic acid sequence (e.g., a DNA sequence) into a host cell without insertion into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In additional embodiments, the DNA construct or vector is co-transformed with a plasmid without being inserted into the plasmid. In a further embodiment, the selectable marker is deleted from the altered Bacillus strain by methods known in the art (see, e.g., Stahl et al., J. Bacteriol. 158:411-418 (1984); and Palmeros et al., Gene 247:255-264 (2000)).

[0154] In some embodiments, the transformed cells are cultured in a conventional nutrient medium. Suitable specific culture conditions, such as temperature, pH, etc., are known to those of skill in the art and are well described in the scientific literature. Some embodiments provide a culture (e.g., a cell culture) that comprises one or more subtilisin variants or nucleic acid sequences described herein.

[0155] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more subtilisin variants described herein are cultured in a suitable nutrient medium under conditions that allow expression of the variants, and the resulting variants are then recovered from the culture. In some embodiments, the variants produced by the cells are recovered from the culture medium by conventional procedures, including, for example, but not limited to, separation of the host cells from the medium by centrifugation or filtration, precipitation of proteinaceous components of the supernatant or filtrate with a salt (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).

[0156] In some embodiments, one or more subtilisin variants produced by a recombinant host cell are secreted into the culture medium. A nucleic acid sequence encoding a purification-facilitating domain can be used to facilitate purification of the variants. A vector or DNA construct comprising a polynucleotide sequence encoding one or more subtilisin variants described herein may further comprise a nucleic acid sequence encoding a purification-facilitating domain to facilitate purification of the variants (see, e.g., Kroll et al., DNA Cell Biol. 12:441-53 (1993)). Such purification-facilitating domains include, but are not limited to, metal-chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath, Protein Expr. Purif. 3:263-281

[1992] ), protein A domains that allow purification on immobilized immunoglobulins, and domains utilized in the FLAGS extension / affinity purification system. The inclusion of a cleavable linker sequence, such as Factor XA or enterokinase (e.g., those available from Invitrogen, San Diego, Calif.) between the purification domain and the heterologous protein is also used to facilitate purification.

[0157] A wide variety of methods can be used to determine the production levels of one or more of the mature subtilisin variants described herein in a host cell. Such methods include, but are not limited to, methods that utilize either polyclonal or monoclonal antibodies specific for the protease. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), and fluorescence-activated cell sorting (FACS). These and other assays are well known in the art (see, e.g., Maddox et al., J. Exp. Med. 158:1211 (1983)).

[0158] Some other embodiments provide methods for making or producing one or more of the mature subtilisin variants described herein. The mature subtilisin variants do not include a signal peptide or propeptide sequence. Some methods include making or producing one or more of the subtilisin variants described herein in a recombinant bacterial host cell, such as, for example, a Bacillus species cell (e.g., a B. subtilis cell). Other embodiments provide methods for producing one or more of the subtilisin variants described herein, the methods comprising culturing a recombinant host cell comprising a recombinant expression vector comprising a nucleic acid sequence encoding one or more of the subtilisin variants described herein under conditions conducive to production of the variants. Some such methods further comprise recovering the variants from the culture.

[0159] Further embodiments include methods of producing one or more of the subtilisin variants described herein, comprising: (a) introducing into a population of cells (e.g., bacterial cells, such as B. subtilis cells) a recombinant expression vector that includes a nucleic acid encoding the variant; and (b) culturing the cells in a culture medium under conditions that promote production of the variant encoded by the expression vector. Some such methods further include (c) isolating the mutant from the cell or from the culture medium.

[0160] Further embodiments relate to methods of improving the cleaning performance or stability of a subtilisin, comprising modifying the subtilisin to include one or more substitutions, or a combination of substitutions, as provided herein.

[0161] Unless otherwise stated, all ingredient or composition levels refer to the activity level of that ingredient or composition, and exclude impurities such as residual solvents or by-products that may be present in commercial sources. The weight of enzyme ingredients is based on total active protein. All percentages and ratios are calculated by weight unless otherwise stated. All percentages and ratios are calculated based on the total composition unless otherwise stated. The compositions described herein include cleaning compositions, such as detergent compositions. In the exemplified detergent compositions, enzyme levels are expressed by pure enzyme by weight of the total composition, and detergent ingredients are expressed by weight of the total composition unless otherwise specified.

[0162] In one embodiment, one or more subtilisin variants described herein are useful in cleaning applications, including, but not limited to, for example, cleaning dishes or tableware, fabrics, medical instruments, and items having hard surfaces (e.g., tables, table tops, walls, furniture, floors, and ceilings hard surfaces). In other embodiments, one or more subtilisin variants described herein are useful in disinfection applications, including, but not limited to, for example, disinfecting automatic dishwashers or washing machines.

[0163] Another embodiment relates to a composition comprising one or more subtilisin variants described herein. In some embodiments, the composition is a cleaning composition. In other embodiments, the composition is a detergent composition. In still other embodiments, the composition is selected from a laundry detergent composition, an automatic dishwashing (ADW) composition, a hand (manual) dishwashing detergent composition, a hard surface cleaning composition, an eyeglass cleaning composition, a medical instrument cleaning composition, a disinfectant (e.g., malodor or microbial) composition, and a personal care cleaning composition. In still other embodiments, the composition is a laundry detergent composition, an ADW composition, or a hand (manual) dishwashing detergent composition. Still further embodiments relate to fabric cleaning compositions, while other embodiments relate to non-fabric cleaning compositions. In some embodiments, the cleaning composition does not comprise boron. In other embodiments, the cleaning composition does not comprise phosphate. In still other embodiments, the composition comprises one or more subtilisin variants described herein and one or more of excipients, adjuncts, and / or additional enzymes.

[0164] Protease Stabilizers Peptide aldehydes can be used as protease stabilizers in detergent formulations as previously described (WO 199813458, WO 2011036153, US 20140228274). Examples of peptide aldehyde stabilizers are peptide aldehydes, ketones, or halomethyl ketones, which may be "N-capped" with, for example, a ureido, carbamate, or urea moiety, or "double N-capped" with, for example, a carbonyl, ureido, oxiamide, thioureido, dithiooxamide, or thioxamide moiety (EP 2358857(B1)). The molar ratio of these inhibitors to proteases can be 0.1:1 to 100:1, for example, 0.5:1 to 50:1, 1:1 to 25:1, or 2:1 to 10:1. Other examples of protease stabilizers are benzophenone or benzoanilide derivatives, which may contain a carboxyl group (U.S. Pat. No. 7,968,508). The molar ratio of these stabilizers to proteases is preferably in the range of 1:1 to 1000:1, in particular 1:1 to 500:1, particularly preferably 1:1 to 100:1, and most particularly preferably 1:1 to 20:1.

[0165] Complexing Agents For purposes of the present invention, a "complexing agent" is a compound capable of combining with multivalent ions, such as calcium, magnesium, lead, copper, zinc, cadmium, mercury, manganese, iron, aluminum, and other cationic multivalent ions, to form water-soluble complexes. The complexing agent has a logarithmic stability constant ([logK]) for Ca2+ of at least 3. The stability constant, logK, is measured in a solution of ionic strength 0.1 at a temperature of 25°C.

[0166] The composition of the present invention preferably comprises 10% to 50% by weight of the composition of a complexing agent system. The complexing agent system comprises one or more complexing agents selected from the group consisting of methylglycine diacetate (MGDA), citric acid, glutamic acid-N,N-diacetate (GLDA), iminodisuccinic acid (IDS), carboxymethyl inulin, L-aspartic acid N,N-diacetate tetrasodium salt (ASDA) and mixtures thereof. Preferably, the complexing agent system comprises a composition of at least 10% by weight of MGDA. The complexing system may further comprise a complexing agent selected from the group consisting of citric acid, (GLDA), (IDS), carboxymethyl inulin, L-aspartic acid N,N-diacetate tetrasodium salt (ASDA) and mixtures thereof. Preferably, the complexing agent system comprises a composition of at least 10% by weight of MGDA and at least 10% by weight of citric acid. For the purposes of the present invention, the term "acid" when referring to a complexing agent includes acids and their salts.

[0167] In a preferred embodiment, the composition comprises at least 15% by weight of MGDA, more preferably 20% to 40% by weight of the trisodium salt of MGDA. Compositions containing this high level of MGDA perform well in hard water and over long and / or high temperature cycles.

[0168] The complexing agent system of the present invention may further include citric acid.

[0169] Dispersant Polymer The dispersant polymer may be used in any suitable amount from about 0.1 to about 20% by weight of the composition, preferably from 0.2 to about 15% by weight, and more preferably from 0.3 to % by weight.

[0170] The dispersant polymer is capable of suspending calcium or calcium carbonate in the automatic dishwashing process.

[0171] The dispersant polymer has a calcium binding capacity in the range of 30-250 mg Ca / g dispersant polymer, preferably 35-200 mg Ca / g dispersant polymer, more preferably 40-150 mg Ca / g dispersant polymer at 25° C. To determine whether a polymer is a dispersant polymer within the meaning of the present invention, the following calcium binding capacity determination is made according to the instructions below.

[0172] Calcium binding ability test method The calcium binding capacity referred to herein is determined by titration using a pH / ion meter such as a Meettler Toledo SevenMulti™ benchtop meter and a PerfectION™ interdigitated Ca combination electrode. To measure binding capacity, a heating and stirring device suitable for the beaker or tergotometer pot is set to 25° C. and the metered ion electrode is calibrated according to the manufacturer's instructions. Standard concentrations for electrode calibration bracket the test concentrations and must be measured at 25° C. A stock solution of 1000 mg / g Ca is prepared by adding 3.67 g CaCl2-2H2O to 1 L of deionized water, followed by dilutions to prepare three working solutions of 100 mL each containing calcium concentrations of 100 mg / g, 10 mg / g, and 1 mg / g, respectively. The 100 mg Ca / g working solution is used as the initial concentration during titrations performed at 25° C. The ionic strength of each working solution is adjusted by adding 2.5 g / L NaCl, respectively. 100 mL of the 100 mg Ca / g working solution is heated and stirred until it reaches 25°C. An initial measurement of calcium ion concentration is made when the solution reaches 25°C using an ion electrode. Test polymer is then added incrementally (at 0.01 g / L intervals) to the calcium working solution and measured after 5 minutes of stirring after each increment is added. The titration is stopped when the solution reaches 1 mg / g calcium. The titration procedure is repeated with the remaining two calcium concentration working solutions. The binding capacity of the test polymer is calculated as the linear slope of the measured calcium concentration versus grams / L of test polymer added.

[0173] The dispersant polymer preferably has a net negative charge when dissolved in an aqueous solution having a pH greater than 6.

[0174] The dispersant polymer may also have a sulfonated carboxylic acid ester or amide to increase the negative charge at lower pH and improve its dispersing properties in hard water. Preferred dispersant polymers are sulfonated / carboxylated polymers, i.e., polymers that contain both sulfonated and carboxylated monomers.

[0175] Preferably the dispersant polymer is a sulfonated derivative of a polycarboxylic acid and may contain two, three, four or more different monomer units.

[0176] At least one structural unit derived from a carboxylic acid monomer has the general formula (III):

[0177] [ka] wherein R1-R3 are independently selected from hydrogen, methyl, a linear or branched saturated alkyl group having 2-12 carbon atoms, a linear or branched mono- or polyunsaturated alkenyl group having 2-12 carbon atoms, the aforementioned alkyl or alkenyl groups substituted with -NH2 or -OH, or -COOH, or COOR4, and wherein R4 is selected from hydrogen, an alkali metal, or a linear or branched saturated or unsaturated alkyl or alkenyl group having 2-12 carbons; Preferred carboxylic acid monomers include one or more of acrylic acid, maleic acid, maleic anhydride, itaconic acid, citraconic acid, 2-phenylacrylic acid, cinnamic acid, crotonic acid, fumaric acid, methacrylic acid, 2-ethylacrylic acid, methylenemalonic acid, or sorbic acid. Acrylic and methacrylic acid are more preferred.

[0178] Optionally, one or more structural units derived from at least one nonionic monomer have the general formula (IV):

[0179] [ka] wherein R5-R7 are independently selected from hydrogen, methyl, phenyl, or a hydroxyalkyl group containing 1-6 carbon atoms, which may be part of a cyclic structure; X is an optionally present spacer group selected from -CH2-, -COO-, -CONH-, or -CONR8-; and R8 is selected from linear or branched, saturated alkyl groups having 1-22 carbon atoms or unsaturated, preferably aromatic, groups having 6-22 carbon atoms.

[0180] Preferred nonionic monomers are butene, isobutene, pentene, 2-methylpent-1-ene, 3-methylpent-1-ene, 2,4,4-trimethylpent-1-ene, 2,4,4-trimethylpent-2-ene, cyclopentene, methylcyclopentene, 2-methyl-3-methyl-cyclopentene, hexene, 2,3-dimethylhex-1-ene, 2,4-dimethylhex-1-ene, 2,5-dimethylhex-1-ene, 3,5-dimethylhex-1-ene, 4,5-dimethylhex-1-ene, 5,5-dimethylhex-1-ene, 6,5-dimethylhex-1-ene, 7,5-dimethylhex-1-ene, 8,5-dimethylhex-1-ene, 9,5-dimethylhex-1-ene, 10,5-dimethylhex-1-ene, 11,5-dimethylhex-1-ene, 12,5-dimethylhex-1-ene, 13,5-dimethylhex-1-ene, 14,5-dimethylhex-1-ene, 15,5-dimethylhex-1-ene, 16,5-dimethylhex-1-ene, 17,5-dimethylhex-1-ene, 18,5-dimethylhex-1-ene, 19,5-dimethylhex-1-ene, 20,5-dimethylhex-1-ene, 21,5-dimethylhex-1-ene, 22,5-dimethylhex-1-ene, 23,5-dimethylhex-1-ene, 24,5-dimethylhex-1-ene, 25,5-dimethylhex-1-ene, 26,5-dimethylhex-1-ene, 27,5-dimethylhex-1-ene, 28,5-dimethylhex-1-ene, 29,5-dimethylhex-1-ene, 21,5-dimethylhex-1-ene, 22,5-dimethylhex-1-ene, 23,5-dimethylhex- The aromatic monomers include one or more of α-olefins having 10 or more carbon atoms, such as dimethylhex-1-ene, 4,4-dimethylhex-1-ene, cyclohexene, methylcyclohexene, cycloheptene, dec-1-ene, dodec-1-ene, hexadec-1-ene, octadec-1-ene, and docos-1-ene, and preferred aromatic monomers are styrene, α-methylstyrene, 3-methylstyrene, 4-dodecylstyrene, 2-ethyl-4-styrene, 1-ethyl-1 ... Preferred carboxylic acid ester monomers are methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, t-butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate and behenyl (meth)acrylate, and preferred amides are N-methylacrylamide, N-ethylacrylamide, N-t-butylacrylamide, N-2-ethylhexylacrylamide, N-octylacrylamide, N-lauryl acrylamide, N-stearyl acrylamide and N-behenyl acrylamide.

[0181] and at least one structural unit derived from at least one sulfonic acid monomer has the general formulas (V) and (VI):

[0182] [ka] wherein R7 is a group containing at least one sp2 bond, A is O, N, P, S, an amide or ester bond, B is a monocyclic or polycyclic aromatic group or an aliphatic group, each t is independently 0 or 1, and M+ is a cation. In one embodiment, R7 is a C2-C6 alkene. In another embodiment, R7 is ethene, butene, or propene.

[0183] Preferred sulfonated monomers include one or more of 1-acrylamido-1-propanesulfonic acid, 2-acrylamido-2-propanesulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methacrylamido-2-methyl-1-propanesulfonic acid, 3-methacrylamido-2-hydroxy-propanesulfonic acid, allylsulfonic acid, methallyl sulfonic acid, allyloxybenzenesulfonic acid, methallyloxybenzenesulfonic acid, 2-hydroxy-3-(2-propenyloxy)propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, styrenesulfonic acid, vinylsulfonic acid, 3-sulfopropyl, 3-sulfo-propyl methacrylate, sulfomethacrylamide, sulfomethylmethacrylamide, and mixtures of the aforementioned acids or water-soluble salts thereof.

[0184] Preferably, the polymer comprises the following concentrations of monomers: from about 40 to about 90%, preferably from about 60 to about 90%, by weight of the polymer of one or more carboxylic acid monomers, from about 5 to about 50%, preferably from about 10 to about 40%, by weight of the polymer of one or more sulfonic acid monomers, and optionally from about 1% to about 30%, preferably from about 2 to about 20%, by weight of the polymer of one or more non-ionic monomers. Particularly preferred polymers comprise from about 70% to about 80% by weight of the polymer of at least one carboxylic acid monomer and from about 20% to about 30% by weight of the polymer of at least one sulfonic acid monomer.

[0185] In the polymer, all or some of the carboxylic or sulfonic acid groups may be present in neutralized form, i.e. the acidic hydrogen atoms of the carboxylic and / or sulfonic acid groups in some or all of the acidic groups may be replaced by metal ions, preferably alkali metal ions, in particular sodium ions.

[0186] The carboxylic acid is preferably (meth)acrylic acid. The sulfonic acid monomer is preferably 2-acrylamido-2-propanesulfonic acid (AMPS).

[0187] Preferred commercially available polymers include Alcosperse 240, Aquatreat AR540, and Aquatreat MPS supplied by Alco Chemical, Acumer 3100, Acumer 2000, Acusol 587G, and Acusol 588G supplied by Rohm & Haas, Goodrich K-798, K-775, and K-797 supplied by BF Goodrich, and ACP1042 supplied by ISP technologies Inc. Particularly preferred polymers are Acusol 587G and Acusol 588G supplied by Rohm & Haas.

[0188] Suitable dispersant polymers include low molecular weight anionic carboxylic acid polymers. They may be homopolymers or copolymers having a weight average molecular weight of about 200,000 g / mol or less, or about 75,000 g / mol or less, or about 50,000 g / mol or less, or about 3,000 to about 50,000 g / mol, preferably about 5,000 to about 45,000 g / mol. The dispersant polymer may be a low molecular weight homopolymer of polyacrylate having an average molecular weight of 1,000 to 20,000, in particular 2,000 to 10,000, and particularly preferably 3,000 to 5,000.

[0189] The dispersant polymer may be a copolymer of acrylic acid and methacrylic acid, a copolymer of acrylic acid and / or methacrylic acid and maleic acid, and a copolymer of acrylic acid and / or methacrylic acid and fumaric acid, having a molecular weight of less than 70,000, with a molecular weight in the range of 2,000 to 80,000, more preferably 20,000 to 50,000, specifically 30,000 to 40,000 g / mol, and with a ratio of (meth)acrylate to maleate or fumarate segments of 30:1 to 1:2.

[0190] The dispersant polymer may be a copolymer of acrylamide and acrylate having a molecular weight of 3,000 to 100,000, alternatively 4,000 to 20,000, and an acrylamide content of less than 50%, alternatively less than 20%, by weight of the dispersant polymer may also be used. Alternatively, such dispersant polymer may have a molecular weight of 4,000 to 20,000, and an acrylamide content of 0% to 15% by weight of the polymer.

[0191] Suitable dispersant polymers herein also include itaconic acid homopolymers and copolymers.

[0192] Alternatively, the dispersant polymer may be selected from the group consisting of alkoxylated polyalkyleneimines, alkoxylated polycarboxylates, polyethylene glycols, styrene copolymers, cellulose sulfate esters, carboxylated polysaccharides, amphiphilic graft copolymers, and mixtures thereof.

[0193] Bleach-based The compositions of the present invention preferably include a bleaching system comprising a high concentration of bleaching agent, preferably percarbonate, in combination with a bleach activator or a bleach catalyst or both. Preferably, the bleach activator is TAED and the bleach catalyst is a manganese bleach catalyst.

[0194] Bleach The compositions of the present invention preferably comprise from about 10% to about 20%, more preferably from about 12% to about 18%, by weight of the composition, of a bleaching agent, preferably a percarbonate.

[0195] Due to the improved stability of the variants, the composition can include stronger, more aggressive bleaching agents (e.g., higher levels of bleach catalyst can be used). Less stable bleaching agents (e.g., percarbonate particles with less coating) may be used in the composition.

[0196] Inorganic and organic bleaching agents are suitable for use herein. Inorganic bleaching agents include perhydrated salts such as perborates, percarbonates, perphosphates, persulfates and persilicates. Inorganic perhydrated salts are usually alkali metal salts. Inorganic perhydrated salts can be included as crystalline solids without additional protection. Alternatively, the salts can be coated. Suitable coatings include sodium sulfate, sodium carbonate, sodium silicate and mixtures thereof. The coatings can be applied as a mixture applied to the surface or applied in sequential layers.

[0197] Alkali metal percarbonates, especially sodium percarbonate, are the preferred bleaching agents for use herein. The percarbonate is most preferably incorporated into the product in a coated form which provides in-product stability.

[0198] Potassium monopersulfate peroxide is another inorganic perhydrate salt useful herein.

[0199] Typical organic bleaching agents are organic peroxyacids, especially dodecane diperoxyacid, tetradecane diperoxyacid, and hexadecanediperoxyacid. Mono- and diperazelaic acid, mono- and diperbrassylic acid are also suitable herein. Diacyl and tetraacyl peroxides, such as dibenzoyl peroxide and dilauroyl peroxide, are other organic peroxides that can be used in the context of the present invention.

[0200] Further typical organic bleaching agents include peroxyacids, specific examples being the alkylperoxyacids and arylperoxyacids. Preferred representatives are: (a) peroxybenzoic acid and its ring-substituted derivatives, such as alkylperoxybenzoic acids, as well as peroxy-α-naphthoic acid and magnesium monoperphthalate; (b) aliphatic or substituted aliphatic peroxyacids, such as peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid [phthaloiminoperoxyhexanoic acid (PAP)], o-carboxybenzamidoperoxycaproic acid, N-nonenylamidoperadipic acid and N-nonenylamidopersuccinate; and (c) aliphatic and araliphatic peroxydicarboxylic acids, such as 1,12-diperoxycarboxylic acid, 1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid, diperoxyphthalic acid, 2-decyldiperoxybutane-1,4-dioic acid, N,N-terephthaloyldi(6-aminopercaproic acid).

[0201] bleach activator Bleach activators are typically organic peracid precursors that enhance bleaching action during washing at temperatures up to 60° C. Bleach activators suitable for use herein include compounds which, under perhydrolysis conditions, give aliphatic peroxoycarboxylic acids, preferably having 1 to 12 carbon atoms, especially 2 to 10 carbon atoms, and / or optionally substituted perbenzoic acids. Suitable materials have O-acyl and / or N-acyl groups with the number of carbon atoms specified and / or optionally substituted benzoyl groups. Polyacylated alkylenediamines, specifically tetraacetylethylenediamine (TAED), acylated triazine derivatives, specifically 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylated glycolurils, specifically tetraacetylglycoluril (TAGU), N-acylimides, specifically N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates, specifically n-nonanoyl- or isononanoyloxybenzenesulfonate (n- or iso-NOBS), decanoyloxybenzoic acid, Also preferred are carboxylic acid (DOBA), carboxylic anhydrides, particularly phthalic anhydride, acylated polyhydric alcohols, particularly triacetin, ethylene glycol diacetate and 2,5-diacetoxy-2,5-dihydrofuran, and triethylacetyl citrate (TEAC). When present, the compositions of the invention comprise from 0.01 to 5%, preferably from 0.2 to 2%, by weight of the composition of a bleach activator, preferably TAED.

[0202] Bleaching catalyst The compositions herein preferably contain a bleach catalyst, preferably a metal-containing bleach catalyst. More preferably, the metal-containing bleach catalyst is a transition metal-containing bleach catalyst, especially a manganese or cobalt-containing bleach catalyst.

[0203] Preferred bleaching catalysts for use herein include manganese triazacyclononane and related complexes; Co, Cu, Mn and Fe bispyridylamine and related complexes; and pentamine cobalt(III) acetate and related complexes. Particularly preferred bleaching catalysts for use herein are 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN) and 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN). Particularly preferred compositions for use herein are 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN) and / or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN).

[0204] Preferably, the composition of the present invention comprises 0.001 to 0.5%, more preferably 0.002 to 0.1%, more preferably 0.005 to 0.075% by weight of the composition of a bleach catalyst. Preferably, the bleach catalyst is a manganese bleach catalyst.

[0205] Inorganic Builders The compositions of the present invention preferably comprise an inorganic builder. Suitable inorganic builders are selected from the group consisting of carbonates, silicates and mixtures thereof. Sodium carbonate is especially preferred for use herein. Preferably, the compositions of the present invention comprise 5 to 60% sodium carbonate by weight of the composition, more preferably 10 to 50% and especially 15 to 45%.

[0206] Surfactants The surfactant suitable for use herein includes nonionic surfactant, and preferably, the composition does not contain any other surfactant.Traditionally, nonionic surfactant has been used in automatic dishwashing for surface modification purposes, specifically for sheeting purposes to avoid filming and spotting and improve gloss.It has been found that nonionic surfactant can also contribute to preventing soil redeposition.

[0207] Preferably, the compositions of the present invention comprise a nonionic surfactant or nonionic surfactant system, more preferably the nonionic surfactant or nonionic surfactant system has a phase inversion temperature of 40-70°C, preferably 45-65°C, measured at a concentration of 1% in distilled water. By "nonionic surfactant system" herein is meant a mixture of two or more nonionic surfactants. Nonionic surfactant systems are preferred for use herein. They are believed to have improved cleaning and finishing properties and better stability in products than single nonionic surfactants.

[0208] The phase inversion temperature is the temperature below which a surfactant or mixture thereof will preferentially partition into an aqueous phase as oil-swollen micelles and above which a surfactant or mixture thereof will preferentially partition into an oily phase as water-swollen reverse micelles. The phase inversion temperature can be determined visually by identifying the temperature at which turbidity occurs.

[0209] The phase inversion temperature of a non-ionic surfactant or system can be determined as follows: A solution containing 1% of the corresponding surfactant or mixture by weight of the solution is prepared in distilled water. After gently stirring the solution, the phase inversion temperature is analyzed to ensure that the process occurs at chemical equilibrium. The phase inversion temperature is measured in a thermostable bath by immersing the solution in a 75 mm sealed glass test tube. To ensure there are no leaks, the test tube is weighed before and after measuring the phase inversion temperature. The temperature is gradually increased at a rate of less than 1° C. / min until it reaches a few degrees below the pre-estimated phase inversion temperature. The phase inversion temperature is determined visually at the first sign of turbidity.

[0210] Suitable nonionic surfactants include i) ethoxylated nonionic surfactants prepared by reaction of monohydroxyalkanols or alkyphenols having 6 to 20 carbon atoms with, preferably, at least 12 moles, particularly preferably at least 16 moles, and even more preferably at least 20 moles of ethylene oxide per mole of alcohol or alkylphenol, ii) alcohol alkoxylated surfactants having 6 to 20 carbon atoms and at least one ethoxy and propoxy group. Mixtures of surfactants i) and ii) are preferred for use herein.

[0211] Other suitable nonionic surfactants are the epoxy-capped poly(oxyalkylated) alcohols represented by the formula: R1O[CH2CH(CH3)O]x[CH2CH2O]y[CH2CH(OH)R2] (I) In the formula, R1 is a linear or branched aliphatic hydrocarbon group having 4 to 18 carbon atoms, R2 is a linear or branched aliphatic hydrocarbon group having 2 to 26 carbon atoms, x is an integer having an average value of 0.5 to 1.5, more preferably about 1, and y is an integer having a value of at least 15, more preferably at least 20.

[0212] Preferably, the surfactant of formula I has at least about 10 carbon atoms in the terminal epoxide unit [CH2CH(OH)R2]. In accordance with the present invention, a suitable surfactant of formula I is, for example, POLY-TERGENT® SLF-18B nonionic surfactant of Olin Corporation, as described in WO 94 / 22800, published October 13, 1994 by Olin Corporation.

[0213] enzyme Other proteases The compositions of the invention can include proteases in addition to the proteases of the invention. A mixture of two or more proteases can contribute to enhanced cleaning over a broader temperature, cycle duration and / or substrate range, and can provide superior shine benefits, especially when used with anti-redeposition agents and / or sulfonated polymers.

[0214] Suitable proteases for use in combination with the variant proteases of the present invention include metalloproteases and serine proteases, including neutral or alkaline microbial serine proteases, such as subtilisin (EC 3.4.21.62). Suitable proteases include those of animal, vegetable or microbial origin. In one aspect, such suitable proteases may be of microbial origin. Suitable proteases include chemically or genetically modified variants of the aforementioned suitable proteases. In one aspect, suitable proteases may be serine proteases, such as alkaline microbial proteases or / and trypsin-type proteases. Examples of suitable neutral or alkaline proteases include: Subtilisins (EC 3.4.21.62), in particular WO 2004067737, WO 2015091989, WO 2015091990, WO 2015024739, WO 2015143360, U.S. Pat. Nos. 6,312,936(B1), 5,679,630, 4,760,025, WO 03 / 055974, WO 03 / 054185, WO 03 / 054184, WO 2017 / 215925, German Patent Application Publication No. 102006022216(A1), WO 2015089447, Those derived from Bacillus species, such as B. lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, B. gibsonii, and B. akibaii, as described in Nos. 2015089441, 2016066756, 2016066757, 2016069557, 2016069563, 2016069569, 2016174234, 2017 / 089093, 2020 / 156419, and 2016 / 183509. Specifically, the mutations S9R, A15T, V66A, A188P, V199I, N212D, Q239R, N255D, X9E, X200L, X256E, X9R, X19L, X60D (Savinase numbering system); subtilisins derived from B. pumilus, such as those described in DE 102006022224(A1), WO 2020 / 221578, WO 2020 / 221579, WO 2020 / 221580, e.g. mutants containing amino acid substitutions at at least one of the positions selected from 9, 130, 133, 144, 224, 252, 271 (BPN' numbering system). (b) trypsin-type or chymotrypsin-type proteases, such as trypsin (e.g., of porcine or bovine origin), including the Fusarium proteases described in WO 89 / 06270, and the chymotrypsin proteases from Cellulomonas described in WO 05 / 052161 and WO 05 / 052146. (c) metalloproteases, in particular those derived from Bacillus amyloliquefaciens as described in WO 07 / 044993(A2), those derived from Bacillus, Brevibacillus, Thermoactinomyces, Geobacillus, Paenibacillus, Lysinibacillus or Streptomyces species as described in WO 2014194032, WO 2014194054 and WO 2014194117, those derived from Kluyvera aluminosa as described in WO 2015193488, and those derived from Streptomyces and Lysobacter as described in WO 2016075078. (d) proteases having at least 90% identity to the subtilase from Bacillus sp. TY145, NCIMB 40339, as described in WO 92 / 17577 (Novozymes A / S), including variants of this Bacillus sp. TY145 subtilase as described in WO 2015024739 and WO 2016066757.

[0215] Additional proteases that are particularly preferred for the compositions of the invention are variants of a parent protease, which parent protease exhibits at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, in particular 100% identity with SEQ ID NO:2, and which variants comprise one or more, or two or more, or three or more substitutions at the following positions relative to SEQ ID NO:2: S3V, S9R, A13V, A15T, G20 * , L21F, I35V, N60D, V66A, N74D, S85N / R, S97SE, S97AD, S97D / G, S99G / M / D / E, S101A, V102E / I, G116V / R, S126F / L, P127Q, S128A, S154 D, G157S, Y161A, R164S, A188P, V199I, Q200C / E / I / K / T / V / W / L, Y203W, N212D, M216S / F, A222V, Q239R / F, T249R, N255D and L256E / N / Q / D

[0216] Preferred proteases include the following variants: S9R+A13V+A15T+l35V+N60D+Q239F, or S9R+A15T+G20+L21F+N60D+Q239N, or S9R+A15T+V66A+S97G+A222V+Q239R+N255D, or S9R+A15T+V66A+N74D+Q239R, or S9R+A15T+V66A+N212D+Q239R, or S99SE, or S99AD, or N74D+S85R+G116R+S126L+P127Q+S128A, or N74D+S85R+G116R+S126L+P127Q+S128A+S182D+V238R or G116V+S126L+P127Q+S128A or S99M+G116V+S126L+P127Q+S128A, which have at least 90%, preferably at least 95%, identity to SEQ ID NO:2.

[0217] Suitable additional commercially available protease enzymes include those sold by Novozymes under the trade names Alcalase®, Savinase®, Primase®, Durazym®, Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®, Liquanase® Evity®, Savinase® Evity®, Ovozyme®, Neutrase®, Everlase®, Coronase®, Blaze®, Blaze Ultra®, Blaze® Evity®, Blaze® Exceed, Blaze® Pro, Esperase®, Progress® Uno, Progress® Excel, Progress® Key, Ronozyme®, Vinzon®, and Het Ultra®. Sold by A / S (Denmark); those sold by Dupont under the trade names Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®, Excellase®, Ultimase®, and Purafect OXP®; those sold by Solvay Enzymes under the trade names Opticlean® and Optimase®; and those available from Henkel / Kemira, namely, BLAP (the sequence of which is shown in FIG. 29 of U.S. Pat. No. 5,352,604 and which has the mutations S99D+S101R+S103A+V104I+G159S, hereinafter referred to as BLAP), BLAP R (BLAP with S3T+V4I+V199M+V205I+L217D), BLAP and BLAP X (BLAP with S3T+V4I+V205I) and BLAP F49 (BLAP with S3T+V4I+A194P+V199M+V205I+L217D); and optionally at least one further mutation 101E / D, S156D, L262; KAP (Bacillus alkalophilus subtilisin with mutations A230V+S256G+S259N) from Kao; and Lavergy®, Lavergy® Pro, Lavergy® C Bright from BASF.

[0218] Particularly preferred for use herein in combination with the variant proteases of the invention are commercially available proteases selected from the group consisting of Properase®, Blaze®, Ultimase®, Everlase, Savinase®, Savinase Evity®, Savinase Ultra®, Excellase®, Ovozyme®, Coronase®, Blaze Evity® and Blaze Pro®, BLAP and BLAP variants.

[0219] Preferred concentrations of protease in the products of the present invention include from about 0.05 to about 10, more preferably from about 0.5 to about 7, and especially from about 1 to about 6 mg of active protease per gram of composition.

[0220] amylase Preferably, the composition of the invention may comprise an amylase. Suitable α-amylases include those of bacterial or fungal origin. Chemically or genetically modified variants (mutants) are included. Preferred alkaline α-amylases are derived from Bacillus species, such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus sterothermophilus, Bacillus subtilis or other Bacillus species, such as Bacillus species NCBI12289, NCBI12512, NCBI12513, DSM9375 (US Pat. No. 7,153,818), DSM12368, DSMZ No. 12649, KSM AP1378 (WO 97 / 00324), KSM K36 or KSM K38 (EP 1,022,334). Preferred amylases include:

[0221] Suitable amylases are recombinant, non-naturally occurring variants of a parent α-amylase, where the variant α-amylase has 95% identity to SEQ ID NO:5 and has amino acid substitutions at positions 51 and 125 relative to SEQ ID NO:5. The variant α-amylase may have amino acid substitutions T51V and S125R relative to SEQ ID NO:5. The variant α-amylase may further have amino acid substitutions at positions 172, 227 or 231 relative to SEQ ID NO:5. The variant α-amylase may further have amino acid substitutions N172Q, N227R or F231L relative to SEQ ID NO:5.

[0222] One suitable amylase is a recombinant, non-naturally occurring variant of a parent α-amylase, where the variant α-amylase has 95% identity to SEQ ID NO:5 and has the following amino acid substitutions with respect to SEQ ID NO:5: (a) T51V+S125R+F231L; (b) T51V+S125R+N172Q+N227R; (c) N029Q+T051V+T244I+S253L+K268R+K319R+S418A; or (d) E415G,

[0223] Other preferred amylases include the following: (a) variants described in WO 96 / 23873, WO 00 / 60060, WO 06 / 002643 and WO 2017 / 192657, in particular variants having one or more substitutions at the following positions relative to the AA560 enzyme listed in SEQ ID NO: 12 in WO 06 / 002643: 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 202, 214, 231, 246, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482, 484, preferably D183 * and G184 * Mutants that also contain deletions of (b) SEQ ID NO: 4 of WO 06 / 002643, variants exhibiting at least 90% identity to the wild-type enzyme from Bacillus sp. SP722, in particular variants having deletions at positions 183 and 184, as well as variants described in WO 2000 / 60060, WO 2011 / 100410 and WO 2013 / 003659, which are incorporated herein by reference. (c) variants exhibiting at least 95% identity with the wild-type enzyme from Bacillus sp. 707 (SEQ ID NO: 7 in U.S. Pat. No. 6,093,562), in particular those comprising one or more of the following mutations: M202, M208, S255, R172 and / or M261. Preferably, the amylase comprises one or more of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N and / or R172Q. Particularly preferred are those comprising the M202L or M202T mutations. (d) a variant described in WO 09 / 149130, preferably SEQ ID NO: 1 or SEQ ID NO: 2 of WO 09 / 149130, which exhibits at least 90% identity to the wild-type enzyme from Geobacillus Stearophermophilus or a truncated version thereof. (e) A variant exhibiting at least 89% identity to SEQ ID NO: 1 of WO2016091688, in particular one containing a deletion at positions H183+G184 and one or more mutations at positions 405, 421, 422 and / or 428. (f) A variant showing at least 60% amino acid sequence identity with “PcuAmyl α-amylase” derived from Paenibacillus cardanolyticus YK9 (SEQ ID NO: 3 in WO 2014099523). (g) A variant showing at least 60% amino acid sequence identity with "CspAmy2 amylase" from Cytophaga species (SEQ ID NO: 1 in WO 2014164777). (h) A variant exhibiting at least 85% identity to AmyE from Bacillus subtilis (SEQ ID NO: 1 of WO 2009149271). (i) A variant exhibiting at least 90% identity with the wild-type amylase derived from Bacillus sp. KSM-K38 having accession number AB051102. (j) A variant exhibiting at least 90%, preferably at least 95%, preferably at least 98% identity to the mature amino acid sequence of AAI10 from Bacillus species (SEQ ID NO: 7 in WO2016180748). (k) A variant that exhibits at least 80% identity to the mature amino acid sequence of Alicyclobacillus sp. amylase (SEQ ID NO: 8 of WO2016180748).

[0224] Preferably, the amylase is a genetically engineered enzyme, in which one or more of the amino acids susceptible to bleaching oxidation are replaced by less oxidizable amino acids. In particular, it is preferred that the methionine residue is replaced by any other amino acid. In particular, it is preferred that the methionine residue most susceptible to oxidation is replaced. Preferably, the methionine at position corresponding to 202 in the AA560 enzyme listed as SEQ ID NO: 12 in WO 06 / 002643 is replaced. Preferably, the methionine at this position is replaced by threonine or leucine, preferably leucine.

[0225] Suitable commercially available α-amylases include DURAMYL®, LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®, STAINZYME®, STAINZYME PLUS®, FUNGAMYL®, ATLANTIC®, INTENSA® and BAN® (Novozymes A / S, Bagsvaerd, Denmark), KEMZYM® AT 9000 (Biozym Biotech Trading GmbH Wehlistrasse 27b A-1200 Wien Austria), RAPIDASE®, PURASTAR®, ENZYSIZE®, OPTISIZE HT PLUS®, POWERASE®, PREFERENZ S® series (PREFERENZ S1000® and PREFERENZ S2000®) and PURASTAR OXAM® (DuPont., Palo Alto, California) and KAM® (Kao, 14-10 Nihonbashi Kayabacho, 1-chome, Chuo-ku Tokyo 103-8210, Japan). In one aspect, suitable amylases include ATLANTIC®, STAINZYME®, POWERASE®, INTENSA®, and STAINZYME PLUS® and mixtures thereof.

[0226] Preferably, the composition of the present invention contains at least 0.01 mg, preferably about 0.05 to about 10, more preferably about 0.1 to about 6, especially about 0.2 to about 5 mg, of active amylase per gram of composition.

[0227] Preferably, the protease and / or amylase of the composition of the invention is in the form of a granule, the granule comprising more than 29% sodium sulfate by weight of the granule, and / or the sodium sulfate and the active enzyme (protease and / or amylase) are in a weight ratio of 3:1 to 100:1, or preferably 4:1 to 30:1, or more preferably 5:1 to 20:1.

[0228] Crystal Growth Inhibitors Crystal growth inhibitors are materials that can bind to calcium carbonate crystals and prevent the further growth of species such as aragonite and calcite.

[0229] Examples of effective crystal growth inhibitors include phosphonates, polyphosphonates, inulin derivatives, polyitaconic acid homopolymers and cyclic polycarboxylates.

[0230] Suitable crystal growth inhibitors may be selected from the group including HEDP (1-hydroxyethylidene 1,1-diphosphonic acid), carboxymethyl inulin (CMI), tricarballylic acid and cyclic carboxylates. For purposes of the present invention, the term carboxylate encompasses both the anionic and the protonated carboxylic acid forms.

[0231] Cyclic carboxylates contain at least two, preferably three or preferably at least four carboxylate groups, and the cyclic structure is based on either a monocyclic or bicyclic alkane or a heterocycle. Suitable cyclic structures include cyclopropane, cyclobutane, cyclohexane or cyclopentane or cycloheptane, bicyclo-heptane or bicyclo-octane, and / or tetrahydrofuran. One of the preferred crystal growth inhibitors is cyclopentane tetracarboxylate.

[0232] Cyclic carboxylates having at least 75%, and preferably 100%, of the carboxylate groups on the same side of the three-dimensional structure of the ring or in the "cis" position are preferred for use herein.

[0233] It is preferred that the two carboxylate groups on the same side of the ring are directly adjacent or in the "ortho" position.

[0234] Preferred crystal growth inhibitors include HEDP, tricarballylic acid, tetrahydrofuran tetracarboxylic acid (THFTCA) and cyclopentane tetracarboxylic acid (CPTCA). THFTCA is preferably in the 2c,3t,4t,5c configuration and CPTCA is in the cis,cis,cis,cis configuration. A particularly preferred crystal growth inhibitor for use herein is HEDP.

[0235] Partially decarboxylated polyitaconic acid homopolymers are also preferred for use herein, preferably with a level of decarboxylation ranging from 50 mol% to 90 mol%. A particularly preferred polymer for use herein is Itaconix TSI®, supplied by Itaconix.

[0236] The crystal growth inhibitor is preferably present in an amount of from about 0.01 to about 10% by weight of the composition, particularly from about 0.02 to about 5% by weight, especially 0.05 to 3% by weight.

[0237] Metal Care Products The metal care agent can prevent or reduce tarnishing, corrosion or oxidation of metals including aluminum, stainless steel, and non-ferrous metals such as silver and copper. Preferably, the composition of the present invention comprises 0.1-5% by weight of the product, more preferably 0.2-4% by weight, especially 0.3-3% by weight of the metal care agent, and preferably the metal care agent is benzotriazole (BTA).

[0238] Glass care products The glass care agent protects the appearance of glassware during the dishwashing process. Preferably, the composition of the present invention comprises 0.1-5% by weight of the composition, more preferably 0.2-4% by weight, especially 0.3-3% by weight of a metal care agent, preferably the glass care agent is a zinc-containing material, especially hydrozinc earth. Another suitable glass care agent is polyethyleneimine (PEI). A particularly preferred PEI is Lupasol® FG supplied by BASF.

[0239] pH The automatic dishwashing compositions of the present invention preferably have a pH of from about 9 to about 12, more preferably from about 10 to less than about 11.5, especially from about 10.5 to about 11.5, when measured in a 1% weight / volume aqueous solution in distilled water at 20°C.

[0240] Reserve Alkalinity The automatic dishwashing compositions of the present invention preferably have a reserve alkalinity of from about 10 to about 20, more preferably from about 12 to about 18, at pH 9.5, when measured in NaOH with 100 grams of product at 20° C.

[0241] Washing conditions There is a wide variety of wash conditions, including various detergent formulations, wash water volumes, wash water temperatures, and wash time lengths to which one or more subtilisin variants described herein may be exposed. A low detergent concentration system refers to wash water containing less than about 800 ppm of detergent components. A medium detergent concentration system refers to wash water containing from about 800 ppm to about 2000 ppm of detergent components. A high detergent concentration system refers to wash water containing more than about 2000 ppm of detergent components. In some embodiments, the "cold water wash" of the present invention utilizes a "cold water detergent" suitable for washing at temperatures between about 10°C and about 40°C, between about 20°C and about 30°C, or between about 15°C and about 25°C, as well as all other combinations within the range of about 15°C to about 35°C or 10°C to 40°C.

[0242] Water hardness varies from place to place. Hardness is determined by the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) is a measure of the amount of Ca2+ / Mg 2+ Hardness is expressed as a mixture of grains per gallon (gpg). Most water in the United States is hard, but the degree of hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has between 60 and 181 ppm of hardness minerals (you can convert ppm to grains per U.S. gallon by dividing ppm by 17.1).

[0243] [Table 1]

[0244] EMBODIMENTS OF THE PRESENT DISCLOSURE The following are embodiments of the present invention: 1. An automatic dishwashing composition comprising a surfactant and a protease, The automatic dishwashing composition is in the form of a water-soluble unit dose pouch containing a compartment, the compartment containing both a powder component and a gel component; The protease has the following sequence with respect to SEQ ID NO:1: (i) a mutation at position 122, (ii) one or more mutations at positions 126, 127, 128, 211, and 212; A subtilisin variant comprising: The variant has at least 80% identity to the amino acid sequence of SEQ ID NO:1. 2. The composition according to embodiment 1, wherein the composition is in the form of a multi-compartment pouch comprising two or more compartments, preferably three or more compartments. 3. The composition of embodiment 1 or 2, wherein the protease is present in the gel component. 4. The composition of any one of embodiments 1-3, wherein the composition comprises a bleaching agent, and the bleaching agent is present in the powder component. 5. The mutant (i) a mutation at position M122L, (ii) one or more mutations at positions 126, 127, 128, 211, and 212; 5. The composition of any one of embodiments 1 to 4, comprising: 6. The composition according to any one of embodiments 1 to 5, wherein the variant comprises two or more mutations at positions 126, 127, 128, 211 and 212, preferably three or more mutations at positions 126, 127, 128, 211 and 212, preferably four or more mutations at positions 126, 127, 128, 211 and 212, preferably five mutations at positions 126, 127, 128, 211 and 212. 7. The composition according to any one of embodiments 1 to 6, wherein the variant comprises one or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably two or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably three or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably four or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably five mutations selected from S126A, D127E, F128G, M211Q and N212Q. 8. The composition according to any one of the preceding embodiments, wherein the variant comprises the mutations M122L, S126A, D127E, F128G, M211Q and N212Q. 9. The composition of any one of embodiments 1 to 8, wherein the variant comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:1. 10. The composition of any one of embodiments 1-9, wherein the variant is derived from a parent or reference polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO:1. 11. The composition of any one of the preceding embodiments, wherein the composition comprises a manganese bleach catalyst selected from the group consisting of 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN), 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN), and mixtures thereof. 12. The composition comprises an acyltransferase, an amylase, an α-amylase, a β-amylase, an α-galactosidase, an arabinase, an arabinosidase, an arylesterase, a β-galactosidase, a β-glucanase, a carrageenase, a catalase, a cellulase, a chondroitinase, a cutinase, a dispersin, an endoglucanase, an endo-β-mannanase, an exo-β-mannanase, an esterase, an exo-mannanase, a galactanase, a glucoamylase, a hemicellulase, a hexosaminidase, a hyaluronidase, a keratinase, a laccase, a lactase, a ligninase, a lipase, a lipolytic enzyme, a lipoxygenase, a lysozyme, a mannana. 12. The composition of any one of the preceding claims, comprising one or more other enzymes selected from: proteases, metalloproteases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, PETases, phenol oxidases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonase, tannase, transglutaminase, xylan acetyl-esterases, xylanases, and xylosidases; and combinations thereof. 13. The composition of embodiment 12, wherein the one or more enzymes comprise an amylase selected from the group consisting of AA707, AA560, AAI10, SP722, BspAmy24, and CspAmy1, and variants thereof, and combinations thereof. 14. The composition of embodiment 13, wherein the one or more enzymes comprise a recombinant, non-naturally occurring variant of a parent α-amylase, wherein the variant α-amylase has 95% identity to SEQ ID NO:3 and has amino acid substitutions at positions 51 and 125 relative to SEQ ID NO:3. EXAMPLES

[0245] Example 1 Expression of BG46 subtilisin mutants Bacillus gibsonii Bgi02446 wild-type subtilisin (BG46) is provided in SEQ ID NO: 1. In this study, a BG46 subtilisin mutant with substitutions S039E, S099R, S126A, D127E, and F128G (SEQ ID NO: 2) was used as a starting point in engineering further substitution mutants, referred to as BG46+S039E-S099R-S126A-D127E-F128G. All BG46 subtilisin variants were expressed using a DNA fragment containing, in consecutive order, the 5' AprE flanking region including variants of the B. subtilis rrnIp2 promoter sequence (SEQ ID NO:3) (the B. subtilis rrnIp2 promoter and the engineered variants are more fully described in patent application WO2020112609), a nucleotide sequence encoding the aprE signal peptide sequence (SEQ ID NO:4), a nucleotide sequence encoding the B. lentus polypeptide (SEQ ID NO:5), a sequence corresponding to the gene encoding mature BG46 subtilisin, the BPN' terminator (SEQ ID NO:6), and the 3' AprE flanking sequence including the kanamycin resistance gene expression cassette (SEQ ID NO:7). The DNA fragment was assembled using standard molecular biology techniques. The linear DNA of the expression cassette was used to transform competent B. subtilis cells of the appropriate strain.

[0246] The transformation mixture was plated on LA plates containing 1.6% skim milk and 1.8 ppm kanamycin and incubated overnight at 37° C. Single colonies were picked and grown in Luria broth at 37° C. under antibiotic selection.

[0247] For protein expression experiments, transformed cells were grown in 96-well microtiter plates (MTPs) in culture medium (a concentrated semi-defined medium based on MOPS buffer containing urea as the main nitrogen source, glucose as the main carbon source, supplemented with 1% soytone for robust cell growth, and containing antibiotic selection) at 32°C, 300 rpm, and 80% humidity in a shaking incubator for 3 days. After centrifugation and filtration, the clarified culture supernatants containing the proteases of interest were used for the assays.

[0248] Example 2 Assay Protein determination The concentration of BG46 subtilisin variants in culture supernatants was determined by UHPLC using a Zorbax 300 SB-C3 column and a linear gradient of 0.1% trifluoroacetic acid (solution A) and 0.07% trifluoroacetic acid (solution B) in acetonitrile and detection at 220 nm. Culture supernatants were diluted with 10 mM NaCl, 0.1 mM CaCl2, 0.005% Tween®-80 and loaded onto the column. Protein concentrations of samples were calculated using a standard curve of purified parent enzyme.

[0249] Protease activity The protease activity of the BG46 subtilisin mutants was tested by measuring the hydrolysis of the AAPF-pNA synthetic peptide substrate.

[0250] For the AAPF assay, the reagent solutions used were: 100 mM Tris pH 8.6, 10 mM CalCl2, 0.005% Tween®-80 in DMSO (Tris / Ca buffer) and 160 mM suc-AAPF-pNA (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare the working solution, 1 mL of suc-AAPF-pNA stock solution was added to 100 mL of Tris / Ca buffer and mixed. Enzyme samples were added to a microtiter plate (MTP) containing 1 mg / mL of suc-AAPF-pNA working solution and assayed for activity at 405 nm for 3-5 min using a SpectraMax plate reader in kinetic mode at room temperature (RT). Protease activity was expressed as mOD / min.

[0251] Stability assay in Tris-EDTA The stability of the BG46 subtilisin mutants described herein was measured by diluting them in stress buffer and measuring the proteolytic activity of the mutants before and after a heat incubation step using the AAPF assay described above. The temperature and duration of the heat incubation step were chosen such that the reference protease showed approximately 15-30% residual activity. Samples were incubated at 60°C for 5 min in a 384-well thermal cycler. Stability was measured in Tris-EDTA (50 mM Tris pH 9; 5 mM EDTA, 0.005% Tween®-80) buffer conditions. Stability results were calculated as percent (%) remaining activity for each enzyme sample by taking the ratio of mOD / min for stressed conditions to non-stressed conditions and multiplying by 100.

[0252] Example 3 Illustrative Examples

[0253] [Table 2]

[0254] [Table 3]

[0255] The solid and gel phases can be combined as desired. The spatial arrangement of the gel phase, which is liquid after mixing of the components and dimensionally stable within a maximum setting time of 10 minutes, can be predetermined by the spatial arrangement of the solid phase and by commercially available or self-designed molds. A water-soluble package in the form of an open pouch can be produced by deep drawing a PVOH-containing film. A liquid composition can be injected into the aforementioned open cavity, resulting in a gel phase after setting, and the solid phase in the form of a free-flowing solid can then be injected into a pouch containing polyvinyl alcohol. 1.0 g of the liquid non-ionic surfactant Genapol EC 50 can be metered onto the obtained particulate surface. The proportion of non-ionic surfactant can be significantly increased without changing the other phase. The open pouch can be sealed by applying a second film and sealing by heat sealing. The prepared detergent portion can be characterized by an attractive aesthetic.

[0256] Dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Instead, unless otherwise indicated, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 mm" is intended to mean "about 40 mm."

Claims

1. 1. An automatic dishwashing composition comprising a surfactant and a protease, The automatic dishwashing composition is in the form of a water-soluble unit dose pouch containing a compartment, the compartment containing both a powder component and a gel component; The protease has the following structure with respect to SEQ ID NO:1: (i) a mutation at position 122, (ii) one or more mutations at positions 126, 127, 128, 211, and 212; A subtilisin variant comprising: The variant has at least 80% identity to the amino acid sequence of SEQ ID NO:

1.

2. 2. The composition according to claim 1, wherein the composition is in the form of a multi-compartment pouch comprising two or more compartments, preferably three or more compartments.

3. The composition of claim 1 or 2, wherein the protease is present in the gel component.

4. A composition according to any one of claims 1 to 3, wherein the composition comprises a bleaching agent, the bleaching agent being present in the powder component.

5. The mutant is (i) a mutation at position M122L, (ii) one or more mutations at positions 126, 127, 128, 211, and 212; The composition according to any one of claims 1 to 4, comprising:

6. 6. The composition of any one of claims 1 to 5, wherein the variant comprises two or more mutations at positions 126, 127, 128, 211 and 212, preferably three or more mutations at positions 126, 127, 128, 211 and 212, preferably four or more mutations at positions 126, 127, 128, 211 and 212, preferably five mutations at positions 126, 127, 128, 211 and 212.

7. 7. The composition of any one of claims 1 to 6, wherein the mutants comprise one or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably two or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably three or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably four or more mutations selected from S126A, D127E, F128G, M211Q and N212Q, preferably five mutations selected from S126A, D127E, F128G, M211Q and N212Q.

8. The composition according to any one of claims 1 to 7, wherein the mutants include the mutations M122L, S126A, D127E, F128G, M211Q and N212Q.

9. 9. The composition of any one of claims 1 to 8, wherein the variant comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

1.

10. 10. The composition of any one of claims 1 to 9, wherein the variant is derived from a parent or reference polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO:

1.

11. 11. The composition of any one of claims 1 to 10, wherein the composition comprises a manganese bleach catalyst selected from the group consisting of 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN), 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN), and mixtures thereof.

12. The composition may comprise an acyltransferase, an amylase, an α-amylase, a β-amylase, an α-galactosidase, an arabinase, an arabinosidase, an arylesterase, a β-galactosidase, a β-glucanase, a carrageenase, a catalase, a cellulase, a chondroitinase, a cutinase, a dispersin, an endoglucanase, an endo-β-mannanase, an exo-β-mannanase, an esterase, an exo-mannanase, a galactanase, a glucoamylase, a hemicellulase, a hexosaminidase, a hyaluronidase, a keratinase, a laccase, a lactase, a ligninase, a lipase, a lipolytic enzyme, a lipoxygenase, a lysozyme, a mannana.

12. The composition of any one of claims 1 to 11, comprising one or more other enzymes selected from: proteases, metalloproteases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, PETases, phenol oxidases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonase, tannase, transglutaminase, xylan acetyl-esterases, xylanases, and xylosidases; and combinations thereof.

13. 13. The composition of claim 12, wherein the one or more enzymes comprise an amylase selected from the group consisting of AA707, AA560, AAI10, SP722, BspAmy24, and CspAmy1, and variants thereof, and combinations thereof.

14. 14. The composition of claim 13, wherein the one or more enzymes comprise a recombinant, non-naturally occurring variant of a parent α-amylase, wherein the variant α-amylase has 95% identity to SEQ ID NO:3 and has amino acid substitutions at positions 51 and 125 relative to SEQ ID NO:3.

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