Home care compositions comprising amylase

Abstract

The present invention relates to a home care composition comprising a surfactant and an amylase.

Description

[Technical Field]

[0001] The present invention is in the field of home care compositions. In particular, the present invention relates to automatic dishwashing detergent compositions. [Background technology]

[0002] Starch consists of a mixture of amylose (15-30% w / w) and amylopectin (70-85% w / w). Amylose consists of linear chains of α-1,4-linked glucose units with a molecular weight (MW) of about 60,000 to about 800,000. Amylopectin is a branched polymer containing α-1,6-branch points every 24-30 glucose units, and its MW can be as high as 100 million.

[0003] Alpha-amylases hydrolyze starch, glycogen, and related polysaccharides by randomly cleaving internal alpha-1,4-glucosidic bonds. Alpha-amylases, particularly those derived from Bacillus, have been used for a variety of different purposes, including starch liquefaction and saccharification, starch modification in the paper and pulp industry, brewing, baking, and the production of syrups for the food industry, the production of feedstock for fermentation processes, and animal feed to increase digestibility. These enzymes can also be used to remove starchy soils and stains during dishwashing.

[0004] The products produced by starch hydrolysis by α-amylases vary with respect to the number of adjacent glucose molecules. Most commercially available α-amylases produce a range of products from glucose (G1) to maltoheptaose (G7). For reasons that are not entirely clear, α-amylases that produce significant amounts of maltopentaose and maltohexaose are believed to be particularly useful for certain commercial applications, including incorporation into detergent cleaning compositions. Many publications have described variations in maltopentaose / maltohexaose-producing α-amylases, etc. Summary of the Invention [Problem to be solved by the invention]

[0005] Nevertheless, there continues to be a need for more robust and better performing engineered α-amylase molecules. [Means for solving the problem]

[0006] The present invention relates to a home care composition comprising a surfactant and an amylase, wherein the amylase is a recombinant, non-naturally occurring variant of a parent α-amylase, and the variant α-amylase has at least 80% identity, preferably at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97%, preferably at least 98% identity, preferably at least 99% identity to SEQ ID NO: 5, and wherein the variant α-amylase has an amino acid substitution at position 51 and / or 125 relative to SEQ ID NO: 5. [Brief explanation of the drawings]

[0007] [Figure 1-1] FIG. 1 shows an alignment of four α-amylases. [Figure 1-2] FIG. 1 shows an alignment of four α-amylases. [Figure 2] The positions of amino acids 51 and 125 in α-amylase AA2560 are indicated. DETAILED DESCRIPTION OF THE INVENTION

[0008] Home Care Compositions The present invention encompasses home care compositions.

[0009] Home care compositions typically refer to consumer and institutional compositions, including, but not limited to, dishwashing and hard surface cleaning compositions, other cleaning agents and cleaning systems for the care and cleaning of inanimate surfaces, and air care compositions.

[0010] The composition is a home care composition, which typically refers to consumer and institutional compositions, including, but not limited to, dishwashing and hard surface cleaning compositions, other detergents and cleaning systems for the care and cleaning of inanimate surfaces, and other compositions specifically designed for the care and maintenance of the home.

[0011] In particular, the composition is an automatic dishwashing composition.The composition comprises an amylase.

[0012] The composition is typically a cleaning composition. Cleaning compositions and cleaning formulations include any composition suitable for cleaning, bleaching, disinfecting, and / or sterilizing any object, item, and / or surface. Such compositions and formulations include, for example, liquid and / or solid compositions, including cleaning or detergent compositions (e.g., liquid, tablet, gel, bar, granule, and / or solid cleaning or detergent compositions; hard surface cleaning compositions and formulations, such as for glass, wood, ceramic, and metal countertops and windows; carpet cleaners; oven cleaners; dishwashing compositions, including hand or manual dishwashing compositions (e.g., "hand" or "manual" dishwashing detergents) and automatic dishwashing compositions (e.g., "automatic dishwashing detergents")). Single-dose unit forms also find use in the present invention, including, but not limited to, pills, tablets, gel caps, or other single-dose units, such as pre-measured powders or liquids.

[0013] cleaning compositions or cleaning formulations, as used herein, unless otherwise specified, include granular or powdered all-purpose or heavy-duty cleaners, particularly cleaning detergents; liquid, granular, gel, solid, tablet, paste, or unit-dose all-purpose cleaners, particularly so-called high-power liquid (HDL) or high-power dry (HDD) detergent types; hand or manual dishwashing cleaners, including those of the high-foaming type; hand or manual dishwashing, automatic dishwashing, or dishware or tableware cleaners, including various tablet, powder, solid, granular, liquid, gel, and rinse aid types for domestic and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and / or hair rinses for humans and other animals; shower gels and foam baths, and metal cleaners; and cleaning aids such as bleaching aids and "stain remover sticks" or pre-treatment types. In some embodiments, granular compositions are in "compact" form; in some embodiments, liquid compositions are in "concentrated" form.

[0014] The terms "detergent composition" or "detergent formulation" are used in reference to compositions intended for use in a wash medium to clean soiled or dirty items. In some embodiments, detergents of the present disclosure include one or more amylases described herein, in addition to one or more surfactants, transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme stabilizers, calcium, enzyme activators, antioxidants, and / or solubilizers. In some examples, the builder salts are mixtures 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).

[0015] The term "adjunct material" refers to any liquid, solid, or gaseous material included in a cleaning composition other than the amylase, or recombinant polypeptide or active fragment thereof, described herein. In some embodiments, cleaning compositions of the present disclosure include one or more cleaning adjunct materials. Each cleaning adjunct 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 adjunct material is compatible with the amylase enzyme used in the composition.

[0016] The phrases "substantially boron-free composition" or "substantially boron-free detergent" refer to a composition or detergent, respectively, that contains trace amounts of boron, possibly from other composition or detergent ingredients, e.g., less than about 1000 ppm (1 mg / kg or liter = 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 of boron.

[0017] The term "bleaching" refers to the treatment of a material or surface for a sufficient length of time and / or under appropriate pH and / or temperature conditions to result in brightening (i.e., whitening) and / or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, ClO, HO, peracids, NO, etc. Bleaching agents also include enzymatic bleaching agents, such as perhydrolases and arylesterases. Another embodiment relates to a composition comprising one or more amylases described herein and one or more perhydrolases, e.g., as described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0018] The term "washing performance" of a protease (e.g., one or more amylases described herein, or recombinant polypeptides or active fragments thereof) refers to the cleaning contribution of one or more amylases described herein, providing additional cleaning performance to a detergent compared to a detergent that does not have one or more amylases described herein added to the composition. Washing performance is compared under appropriate wash conditions. In some test systems, other relevant factors such as detergent composition, suds concentration, water hardness, wash mechanism, time, pH, and / or temperature can be controlled to mimic the condition(s) typical for household use in a particular market segment (e.g., hand or manual dishwashing, automatic dishwashing, tableware washing, tableware washing, etc.).

[0019] The phrase "relevant washing conditions" is used herein to refer to the conditions actually used in homes in the hand dishwashing and automatic dishwashing detergent market segments, in particular washing temperature, time, washing machine, foam concentration, detergent type, and water hardness.

[0020] The term "dishwashing" refers to both domestic and industrial dishwashing, and relates to both automatic dishwashing (eg, in a dishwasher) and manual dishwashing (eg, by hand).

[0021] The term "sanitize" refers to the removal of contaminants from a surface as well as the inhibition or killing of microorganisms on the surface of an item.

[0022] The term "compact" form of the cleaning compositions herein is best reflected by density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are traditional ingredients of powdered detergent compositions. In traditional detergent compositions, filler salts are present in significant amounts, typically about 17 to about 35% by weight of the total composition. In contrast, in compact compositions, filler salts are present in amounts not exceeding about 15% by weight of the total composition. In some embodiments, the filler salts are present in amounts not exceeding about 10% by weight, or more preferably about 5% by weight, of the composition. In some embodiments, the inorganic filler salts are selected from alkali and alkaline earth metal sulfates and chlorides. In some embodiments, the filler salt is sodium sulfate.

[0023] amylase Typically, the compositions and methods of the present invention relate to variant maltopentaose / maltohexaose-forming amylase polypeptides and methods of use thereof. Aspects and embodiments of the compositions and methods of the present invention are summarized in the following separately numbered paragraphs.

[0024] Recombinant, non-naturally occurring variants of a parent α-amylase are provided, wherein the variant α-amylase has at least 80% identity, preferably at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, or preferably at least 99% identity to SEQ ID NO:5, and wherein the variant has an amino acid substitution at position 51 and / or 125 relative to SEQ ID NO:5.

[0025] The variant α-amylase may have amino acid substitutions at positions 51 and 125 relative to SEQ ID NO:5.

[0026] The variant α-amylase may have the amino acid substitutions T51V and / or S125R with respect to SEQ ID NO:5.

[0027] The variant α-amylase may have the amino acid substitutions T51V and S125R with respect to SEQ ID NO:5.

[0028] The variant α-amylase may further comprise one or more, or two or more amino acid substitutions at positions 172, 227 and / or 231 relative to SEQ ID NO:5.

[0029] The variant α-amylase may further comprise amino acid substitutions at positions 172, 227 and 231 relative to SEQ ID NO:5.

[0030] The variant α-amylase may further comprise one or more, or two or more of the amino acid substitutions N172Q, N227R and / or F231L with respect to SEQ ID NO:5.

[0031] The variant α-amylase may further comprise the amino acid substitutions N172Q, N227R and F231L with respect to SEQ ID NO:5.

[0032] The variant α-amylase may comprise the amino acid substitutions (a) T51V+S125R+F231L; (b) T51V + S125R + N172Q + N227R; or (c)N29Q+T51V+S125R+N227R+S253L+G272E+K319R+S418A, may have

[0033] Compositions and methods relating to variant maltopentaose / maltohexaose-forming amylase enzymes are described. The variants were discovered through various experimental approaches, detailed in the accompanying examples. Exemplary uses of the variant amylase enzymes are for cleaning starchy soils in dishwashing and other applications, starch liquefaction and saccharification, improving digestibility in animal feed, and baking and brewing. These and other aspects of the compositions and methods are described in detail below.

[0034] The terms "α-amylase" or "amylolytic enzyme" or amylase in general refer to enzymes that are capable of, among other things, catalyzing the degradation of starch. α-Amylases are hydrolases that cleave α-D-(1→4) O-glycosidic bonds in starch. Generally, α-amylases (EC 3.2.1.1; α-D-(1→4)-glucan glucanohydrolase) are defined as endo-acting enzymes that randomly cleave α-D-(1→4) O-glycosidic bonds in starch molecules to produce polysaccharides containing three or more (1-4)-α-linked D-glucose units. In contrast, some product-specific α-amylases, such as exo-acting amylolytic enzymes (β-amylase (EC 3.2.1.2; α-D-(1→4)-glucan maltohydrolase) and maltogenic α-amylase (EC 3.2.1.133), cleave polysaccharide molecules from the non-reducing end of the substrate. Product-specific amylases, such as β-amylase, α-glucosidase (EC 3.2.1.20; α-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; α-D-(1→4)-glucan glucohydrolase), and maltotetraosidase (EC 3.2.1.60) and maltohexaosidase (EC 3.2.1.98), can produce maltooligosaccharides of specific lengths or concentrated syrups of specific maltooligosaccharides. Some bacterial α-amylases primarily produce maltotetraose (G4), maltopentaose (G5), or maltohexaose (G6) from starch and related α-1,4-glucans, while most α-amylases further convert them to glucose and / or maltose as end products. G6 amylases, such as the AA560 amylase from Bacillus sp. DSM 12649 (i.e., the parent of STAINZYME™) and the Bacillus sp. 707 amylase (also referred to as maltohexaose-forming α-amylase (EC 3.2.1.98)), although technically exo-acting, have a similar structure compared to α-amylases and, in some cases, appear to respond to some of the same beneficial mutations.

[0035] As used herein, "enzyme unit" refers to the amount of product formed per hour under the specified conditions of the assay. For example, a "glucoamylase activity unit" (GAU) is defined as the amount of enzyme that produces 1 g of glucose per hour from a soluble starch substrate (4% DS) at 60°C and pH 4.2. A "soluble starch unit" (SSU) is the amount of enzyme that produces 1 mg of glucose per minute from a soluble starch substrate (4% DS) at pH 4.5 and 50°C. DS refers to "dry solids."

[0036] The term "starch" means a starch of the formula (CH 10 O5) x where X can be any integer. This term refers to any material composed of complex polysaccharide carbohydrates of plants, composed of amylose and amylopectin. This term includes plant materials such as grains, cereals, grasses, tubers, and roots, more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, milo, potato, sweet potato, and tapioca. The term "starch" includes granular starch. The term "granular starch" refers to raw or uncooked starch, e.g., ungelatinized starch.

[0037] As used herein, the terms "liquefaction" or "liquefy" refer to the process of converting starch into less viscous, shorter chain dextrins.

[0038] The terms "wildtype," "parent," or "reference" with respect to a polypeptide refer to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. Similarly, the terms "wildtype," "parent," or "reference" with respect to a polynucleotide refer to a naturally occurring polynucleotide that does not contain an artificial nucleoside change. Note, however, that a polynucleotide encoding a wildtype, parent, or reference polypeptide is not limited to naturally occurring polynucleotides, but encompasses any polynucleotide that encodes a wildtype, parent, or reference polypeptide.

[0039] Reference to a wild-type polypeptide is understood to include the mature form of the polypeptide. A "mature" polypeptide or variant thereof is one in which the signal sequence is absent, e.g., one that is cleaved from the immature form of the polypeptide during or after expression of the polypeptide.

[0040] The term "variant" with respect to a polypeptide refers to a polypeptide that differs from a specific wild-type, parent, or reference polypeptide in that it contains one or more naturally occurring or artificial substitutions, insertions, or deletions of amino acids. Similarly, the term "variant" with respect to a polynucleotide refers to a polynucleotide that differs in nucleotide sequence from a specific wild-type, parent, or reference polynucleotide. The identity of the wild-type, parent, or reference polypeptide or polynucleotide will be clear from the context.

[0041] In the case of the α-amylases of the present invention, "activity" refers to α-amylase activity, which can be measured as described herein.

[0042] The term "performance benefit" refers to an improvement in a desirable property of a molecule. Exemplary performance benefits include, but are not limited to, increased hydrolysis of starch substrates, increased liquefaction performance of grains, cereals, or other starch substrates, increased cleaning performance, increased thermal stability, increased detergent stability, increased storage stability, increased solubility, altered pH profile, reduced calcium dependency, increased specific activity, altered substrate specificity, altered substrate binding, altered pH-dependent activity, altered pH-dependent stability, increased oxidative stability, and increased expression. In some cases, performance benefits are realized at relatively low temperatures. In some cases, performance benefits are realized at relatively high temperatures.

[0043] The terms "protease" and "proteinase" refer to enzyme proteins capable of "proteolysis" or "protein cleavage," which refers to the hydrolysis of the peptide bonds that link amino acids together in the peptide or polypeptide chains that form proteins. This activity of proteases as protein-digesting enzymes is called "proteolytic activity."

[0044] The term "serine protease" refers to enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid in the enzyme's active site. Serine proteases are classified into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like. Serine proteases, particularly subtilisins, are most commonly used in dishwashing detergents.

[0045] "Combinatorial variants" are variants that contain two or more mutations, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, deletions, and / or insertions.

[0046] The term "recombinant," when used with reference to a subject cell, nucleic acid, protein, or vector, indicates that the subject has been altered from its natural state. Thus, for example, a recombinant cell expresses a gene not found in the native (non-recombinant) form of the cell, or expresses a native gene at a different level or under different conditions than found in nature. A recombinant nucleic acid differs from the native sequence by one or more nucleotides and / or is operably linked to a heterologous sequence, e.g., a heterologous promoter in an expression vector. A recombinant protein may differ from the native sequence by one or more amino acids and / or may be fused to a heterologous sequence. A vector containing a nucleic acid encoding an amylase is a recombinant vector.

[0047] The terms "recovered," "isolated," and "separated" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component with which it is naturally associated as found in nature. Such an "isolated" polypeptide includes, but is not limited to, a culture medium containing a secreted polypeptide expressed in a heterologous host cell.

[0048] The term "purified" refers to a material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

[0049] The term "enriched" refers to material (e.g., an isolated polypeptide or polynucleotide) that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.

[0050] The terms "thermostable" and "thermostability," in reference to an enzyme, refer to the ability of the enzyme to retain activity after exposure to elevated temperatures. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (t), given in minutes, hours, or days, during which half of the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring the residual α-amylase activity after exposure to (i.e., exposure by) elevated temperatures.

[0051] "pH range" with respect to an enzyme refers to the range of pH values ​​over which the enzyme exhibits catalytic activity.

[0052] The terms "pH-stable" and "pH stability" in reference to an enzyme refer to the ability of the enzyme to retain activity over a wide range of pH values ​​for a given period of time (eg, 15 minutes, 30 minutes, 1 hour).

[0053] The term "amino acid sequence" is synonymous with, and is used interchangeably with, the terms "polypeptide," "protein," and "peptide." If such an amino acid sequence exhibits activity, it may be referred to as an "enzyme." Conventional one-letter or three-letter codes for amino acid residues are used, and amino acid sequences are presented in the standard amino-carboxy terminal orientation (i.e., N→C).

[0054] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single- or double-stranded and may contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the compositions and methods of the present invention encompass nucleotide sequences that encode specific amino acid sequences. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' orientation.

[0055] A "synthetic" molecule is produced not by an organism, but by in vitro chemical or enzymatic synthesis.

[0056] The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection," "transformation," or "transduction," as known in the art.

[0057] A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts made from cells.

[0058] The term "heterologous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in the host cell.

[0059] The term "endogenous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that is naturally present in a host cell.

[0060] The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence, including both transcription and translation.

[0061] A "signal sequence" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal sequence, which is cleaved during the secretion process.

[0062] "Biologically active" refers to a sequence that has a particular biological activity, for example, enzymatic activity.

[0063] The term "specific activity" refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specified conditions. Specific activity is generally expressed as units (U) / mg protein.

[0064] As used herein, "water hardness" is a measure of the minerals (e.g., calcium and magnesium) present in water.

[0065] "Cultured cell material containing amylase" or similar terms refers to a cell lysate or supernatant (including culture medium) that contains amylase as a component. The cellular material may be from a heterologous host grown in culture to produce amylase.

[0066] "Percent sequence identity" means that a particular sequence has at least a specified percentage of identical amino acid residues in a specified reference sequence when aligned using a software program such as the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are as follows:

[0067] [Table 1]

[0068] Deletions are counted as non-identical residues compared to the reference sequence.

[0069] The term "dry solids" (ds) refers to the total solids of the slurry on a dry weight percent basis. The term "slurry" refers to an aqueous mixture containing insoluble solids.

[0070] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a biochemical production process in which a microorganism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present in the same process step. SSF involves the simultaneous hydrolysis of a starch substrate (granular, liquefied, or solubilized) to sugars, including glucose, and the fermentation of the sugars to alcohol or other biochemicals or biomaterials in the same reaction vessel.

[0071] "Ethanol-producing microorganism" refers to a microorganism that has the ability to convert sugars or oligosaccharides into ethanol.

[0072] The term "fermented beverage" refers to any beverage produced by a method that includes a fermentation process, such as microbial fermentation, for example bacterial and / or fungal fermentation.

[0073] The term "malt" refers to any malted grain, such as malted barley or malted wheat.

[0074] The term "mash" refers to an aqueous slurry of any starch- and / or sugar-containing plant material, such as milling grain, containing, for example, broken barley malt, broken barley, and / or other adjuvants or combinations thereof, which is subsequently mixed with water and separated into wort and spent grain.

[0075] The term "wort" refers to the unfermented liquid that flows out after the milling grains have been extracted during the mash.

[0076] The term "about" refers to ±15% of the reference value.

[0077] 2. Maltopentaose / maltohexaose-forming α-amylase mutants Combinatorial variants of maltopentaose / maltohexaose-forming α-amylases are described that exhibit high performance in automatic dishwashing (ADW) applications. The variants, referred to herein as AA2560, are most closely related to the α-amylase from Bacillus species previously identified as BspAmy24 (SEQ ID NO: 1) in WO 2018 / 184004. The mature amino acid sequence of the AA2560 α-amylase is shown below as SEQ ID NO: 1. HHNGTNGTMM QYFEWHLPND GQHWNRLRND AANLKNLGIT AVWIPPAWKG TSQNDVGYGA YDLYDLGEFN QKGTIRTKYG TRSQLQSAIA SLQNNGIQVY GDVVMNHKGG ADGTEWVQAV EVNPSNRNQE VTGEYTIEAW TKFDFPGRGN THSSFKWRWY HFDGTDWDQS RQLNNRIYKF RGTGKAWDWE VDTENGNYDY LMYADVDMDH PEVINELRRW GVWYTNTLNL DGFRIDAVKH IKYSFTRDWL NHVRSTTGKN NMFAVAEFWK NDLGAIENYL HKTNWNHSVF DVPLHYNLYN ASKSGGNYDM RQILNGTVVS KHPIHAVTFV DNHDSQPAEA LESFVEAWFK PLAYALILTR EQGYPSVFYG DYYGIPTHGV AAMKGKIDPI LEARQKYAYG TQHDYLDHHN IIGWTREGNS AHPNSGLATI MSDGPGGSKW MYVGRHKAGQ VWRDITGNRT GTVTINADGW GNFSVNGGSV SIWVNK

[0078] A closely related maltopentaose / maltohexaose forming α-amylase is from Bacillus sp. 707, designated herein as "AA707." The mature amino acid sequence of AA707 α- is shown below as SEQ ID NO:2. HHNGTNGTMM QYFEWYLPND GNHWNRLNSD ASNLKSKGIT AVWIPPAWKG ASQNDVGYGA YDLYDLGEFN QKGTVRTKYG TRSQLQAAVT SLKNNGIQVY GDVVMNHKGG ADATEMVRAV EVNPNNRNQE VTGEYTIEAW TRFDFPGRGN THSSFKWRWY HFDGVDWDQS RRLNNRIYKF RGHGKAWDWE VDTENGNYDY LMYADIDMDH PEVVNELRNW GVWYTNTLGL DGFRIDAVKH IKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLQ KTNWNHSVFD VPLHYNLYNA SKSGGNYDMR NIFNGTVVQR HPSHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTRE QGYPSVFYGD YYGIPTHGVP AMRSKIDPIL EARQKYAYGK QNDYLDHHNI IGWTREGNTA HPNSGLATIM SDGAGGSKWM FVGRNKAGQV WSDITGNRTG TVTINADGWG NFSVNGGSVS IWVNK

[0079] Another closely related maltopentaose / maltohexaose-forming α-amylase is from Bacillus sp. and is designated AA560. The mature amino acid sequence of AA560 is shown below as SEQ ID NO:3. HHNGTNGTMM QYFEWYLPND GNHWNRLRSD ASNLKDKGIS AVWIPPAWKG ASQNDVGYGA YDLYDLGEFN QKGTIRTKYG TRNQLQAAVN ALKSNGIQVY GDVVMNHKGG ADATEMVRAV EVNPNNRNQE VSGEYTIEAW TKFDFPGRGN THSNFKWRWY HFDGVDWDQS RKLNNRIYKF RGDGKGWDWE VDTENGNYDY LMYADIDMDH PEVVNELRNW GVWYTNTLGL DGFRIDAVKH IKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLN KTNWNHSVFD VPLHYNLYNA SKSGGNYDMR QIFNGTVVQR HPMHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTRE QGYPSVFYGD YYGIPTHGVP AMKSKIDPIL EARQKYAYGR QNDYLDHHNI IGWTREGNTA HPNSGLATIM SDGAGGNKWM FVGRNKAGQV WTDITGNRAG TVTINADGWG NFSVNGGSVS IWVNK

[0080] Based on amino acid sequence identity, another putative maltopentaose / maltohexaose-forming α-amylase is from another Bacillus sp. and is designated herein as AAI10. The mature amino acid sequence of the AAI10 α-amylase is shown below as SEQ ID NO:4. HHDGTNGTIM QYFEWNVPND GQHWNRLHNN AQNLKNAGIT AIWIPPAWKG TSQNDVGYGA YDLYDLGEFN QKGTVRTKYG TKAELERAIR SLKANGIQVY GDVVMNHKGG ADFTERVQAV EVNPQNRNQE VSGTYQIEAW TGFNFPGRGN QHSSFKWRWY HFDGTDWDQS RQLANRIYKF RGDGKAWDWE VDTENGNYDY LMYADVDMDH PEVINELNRW GVWYANTLNL DGFRLDAVKH IKFSFMRDWL GHVRGQTGKN LFAVAEYWKN DLGALENYLS KTNWTMSAFD VPLHYNLYQA SNSSGNYDMR NLLNGTLVQR HPSHAVTFVD NHDTQPGEAL ESFVQGWFKP LAYATILTRE QGYPQVFYGD YYGIPSDGVP SYRQQIDPLL KARQQYAYGR QHDYFDHWDV IGWTREGNAS HPNSGLATIM SDGPGGSKWM YVGRQKAGEV WHDMTGNRSG TVTINQDGWG HFFVNGGSVS VWVKR

[0081] An alignment of these four α-amylases is shown in Figure 1. The amino acid sequence identities are summarized in Table 1. AA707, AA560, and AAI10 all share more than 80% amino acid identity with AA2560.

[0082] [Table 2]

[0083] A variant of the AA2560 α-amylase described in WO 2021 / 080948 that showed excellent cleaning performance is shown below as SEQ ID NO: 5. HHNGTNGTMM QYFEWHLPND GQHWNRLRND AANLKNLGIN AVWIPPAWKG TSQNDVGYGA YDLYDLGEFN QKGTIRTKYG TRSQLQSAIA RLQNNGIQVF GDVVMNHKGG ADGTERVQAV EVNPSNRNQE VTGEYTIEAW TKFDFPGRGN THSSFKWRWY HFDGTDWDQS RNLNNRIYKF TGKAWDWEVD TENGNYDYLM YADVDMDHPE VINELRRWGV WYTNTLNLDG FRIDAVKHIK YQFTRDWLNH VRSTTGKNNM FAVAEFWKND LGAIENYLSK TNWNHSVFDV PLHYNLYNAS KSGGNYDMRQ ILNGTVVSKH PIHAVTFVDN HDSQPAEALE SFVEAWFKPL AYALILTREQ GYPSVFYGDY YGIPTHGVAA MKGKIDPILE ARQKYAYGTQ HDYLDHHNII GWTREGNSAH PNSGLATIMS DGPGGSKWMY VGRHKAGQVW RDITGNRTGT VTINADGWGN FSVNGGSVSI WVNK

[0084] The mutant has the mutations T40N, S91R, Y100F, W116R, Q172N, ΔR181, ΔG182, S244Q and H281S with respect to AA2560 α-amylase, using the wild-type AA2560 α-amylase (SEQ ID NO: 1) for numbering.

[0085] Using the previously described mutant AA2560 α-amylase as a starting point, we engineered additional mutant AA2560 α-amylases that exhibit improved cleaning performance. Most of the new mutants contain two mutations, T51V and S125R. Mutations at these positions result in the loss of hydroxyl groups within the starch-binding groove of the molecule. In the structural model of the enzyme, the hydroxyl groups at T51 and S125 are solvent-exposed and available for hydrogen bonding within the starch-binding groove (Figure 1).

[0086] Without being limited by theory, we propose that the combination of the T51V and S125R mutations may together help reduce non-productive binding modes of starch in the active site by removing hydroxyl groups that, if present, would be exposed for hydrogen bonding within the starch-binding groove. The loss of these hydroxyl groups may prevent starch from binding in a conformation that is incompatible with the optimal placement of the molecule with respect to nucleophiles and general acid / base side chains for catalysis. Based on this theory, other substitutions that remove hydroxyl groups at these positions may provide similar cleaning benefits; therefore, substitutions can be more generally described as T51X and S125X, where X is not S or T.

[0087] Another feature of the variants of the invention follows a mutation at position 91 and / or at least one mutation at the bottom of the α-amylase TIM barrel structure. Barrel bottom residues have a solvent-accessible surface area greater than zero and are located within or adjacent to the core β-barrel structure, on the side of the barrel opposite the active site and on the side containing the N-terminus of each chain. Relevant residues are at positions 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325, 362, 363, and 364, with reference to the numbering in SEQ ID NO: 1. In all cases, the residues line the bottom of the TIM barrel structure, which represents a key structural feature of α-amylases and many other enzymes. An exemplary mutation at residue 91 is the substitution of a polar residue with a charged residue, specifically a positively charged residue such as arginine (ie, X91R), and in the case of AA2560, the specific substitution S91R.

[0088] The variant may further be characterized by mutations within the loop containing surface-exposed residues 167, 169, 171, 172, and 176, with reference to the numbering in SEQ ID NO: 1. The variant may further be characterized by mutations at positions 116 and 281, which are believed to affect solubility.

[0089] The variants may further feature stabilizing mutations at positions 190 and / or 244, with reference to the numbering of SEQ ID NO: 1. Such mutations have been well characterized and are included in currently commercially available α-amylases used for cleaning. Exemplary mutations at these residues are the substitutions X190P and X244A, E, or Q, specifically E190P, S244A, S244E, and S244Q. Mutations at positions 275 and 279 are also of interest in combination with the mutation at position 190.

[0090] The variants may further be characterized by mutations at positions 1, 7, 118, 195, 202, 206, 321, 245, and 459, with reference to the numbering of SEQ ID NO: 1, which are contained in current commercially available α-amylases or proposed for such uses.

[0091] The variants further comprise deletions in the X1G / S1X2G2 motif adjacent to the calcium-binding loop, corresponding to R181, G182, T183, and G184, using SEQ ID NO: 1 for numbering. In some embodiments, the mutant α-amylase comprises adjacent pairwise deletions of amino acid residues corresponding to R181 and G182, or T183 and G184. The deletion at amino acid residues corresponding to R181 and G182 may be referred to as "ΔRG," and the deletion of the amino acid residue corresponding to residue 183 (usually T, D, or H) and G184 may be referred to as "ΔTG," "ΔDG," "ΔHG," etc., as appropriate. Both pairwise deletions appear to produce the same effect in the α-amylase.

[0092] Variants may further include the above mutations for use in (i) any of the well-known Bacillus α-amylases, for example, from B. lichenifomis (i.e., BLA and LAT), B. stearothermophilus (i.e., BSG), and B. amyloliquifaciens (i.e., P00692, BACAM, and BAA), or hybrids thereof; (ii) any α-amylase classified as a Carbohydrate-Active Enzyme Database (CAZy) Family 13 α-amylase; or (iii) other α-amylases that share a similar fold and / or 60% or greater amino acid sequence identity with any amylase traditionally referred to as "Termamyl-like." Exemplary α-amylases include, but are not limited to, those from Bacillus sp. SG-1, Bacillus sp. 707, and the α-amylases designated A7-7, SP722, DSM9014, and KSM AP1378. Similarly, any of the combinations of mutations described herein may provide performance advantages in these α-amylases, whether or not they are described as maltopentaose / maltohexaose producing α-amylases.

[0093] Specifically contemplated combinatorial mutants are listed below with respect to SEQ ID NO: 5, using SEQ ID NO: 5 for numbering. Note that the SEQ ID NO: 5 mutant already has the deletions ΔR181 and ΔG182, thus reducing the number of each position after 183 by two.

[0094] It will be understood that if an α-amylase naturally possesses a mutation listed above (i.e., if the wild-type α-amylase already contains the residue identified as a mutation), that particular mutation does not apply to that molecule, however, other described mutations may work in combination with the naturally occurring residue at that position.

[0095] The variant α-amylases may also contain one or more amino acid substitutions, deletions, or additions in the amino acid sequence, for example, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, or even fewer than 2 substitutions, deletions, or additions. Such variants are expected to have similar activity to the α-amylase from which they are derived. The variant α-amylases may also contain small deletions and / or extensions of one or several residues at their N- or C-termini. Such minor changes are unlikely to detract from the inventive concepts described herein.

[0096] The amylases may be "precursor," "immature," or "full-length" (in which case they include a signal sequence), or "mature" (in which case they lack a signal sequence). The mature forms of the polypeptides are generally most useful. Unless otherwise specified, the numbering of amino acid residues used herein refers to the mature form of the respective amylase polypeptide.

[0097] In some embodiments, the variant α-amylase has at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but less than 100%, amino acid sequence identity to SEQ ID NO:1, 2, 3, 4 or 5, preferably SEQ ID NO:5.

[0098] 2.5. Nucleotides Encoding Variant Amylase Polypeptides In another aspect, nucleic acids encoding variant α-amylase polypeptides are provided. The nucleic acids may encode a particular amylase polypeptide or an α-amylase having a particular degree of amino acid sequence identity to a particular α-amylase.

[0099] In some embodiments, the nucleic acid encodes an α-amylase having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but less than 100%, amino acid sequence identity to SEQ ID NO: 1, 2, 3, or 4 or 5. It is understood that due to the degeneracy of the genetic code, multiple nucleic acids may encode the same polypeptide.

[0100] In some embodiments, the nucleic acid hybridizes under stringent or very stringent conditions to a nucleic acid encoding (or complementary to) an α-amylase having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity to SEQ ID NO: 1, 2, 3, 4, or 5, but less than 100%.

[0101] 3. Production of Mutant α-amylase The variant α-amylases of the invention can be produced in host cells using methods well known in the art, such as by secretion or intracellular expression. Fermentation, separation, and concentration techniques are well known in the art, and conventional methods can be used to prepare concentrated variant α-amylase polypeptide-containing solutions.

[0102] For production-scale recovery, the variant α-amylase polypeptide can be concentrated or partially purified, as generally described above, by removing cells via polymer flocculation. Alternatively, the enzyme can be concentrated or purified by microfiltration, followed by ultrafiltration using available membranes and equipment. However, in some applications, the enzyme does not need to be concentrated or purified, and the entire broth culture can be dissolved and used without further processing. The enzyme can then be processed, for example, into granules.

[0103] Automatic dishwashing compositions Automatic dishwashing compositions can be in any physical form. They may be loose powders, gels, or presented in unit dose form. Preferably, the compositions are in unit dose form, including compressed tablets and water-soluble packs. Automatic dishwashing compositions of the present invention are preferably presented in unit dose form and can be in any physical form, including solid, liquid, and gel forms. The compositions of the present invention are well suited for presentation in multi-compartment packs, more particularly, multi-compartment packs containing compartments with compositions in different physical forms, for example, a compartment containing a solid composition and another compartment containing a liquid composition. The compositions are preferably encapsulated in a water-soluble film such as polyvinyl alcohol. Unit dose compositions enclosed in a polyvinyl alcohol film less than 100 μm thick, preferably 20-90 μm thick, are particularly preferred. The detergent compositions of the present invention weigh from about 8 to about 25 grams, preferably from about 10 to about 20 grams. This weight range allows for a comfortable fit in a dishwasher dispenser. This range represents a small amount of detergent, but the detergent is formulated to provide all of the benefits described herein above.

[0104] The composition is preferably phosphate-free. By "phosphate-free" herein is understood the composition contains less than 1% phosphate by weight of the composition, preferably less than 0.1% phosphate.

[0105] Complexing Agents For purposes of this 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.

[0106] The compositions of the present invention preferably comprise a complexing agent system at 10% to 50% by weight of the composition. 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-diacetic acid (GLDA), iminodisuccinic acid (IDS), carboxymethyl inulin, L-aspartic acid-N,N-diacetic acid tetrasodium salt (ASDA), and mixtures thereof. Preferably, the complexing agent system comprises 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-diacetic acid tetrasodium salt (ASDA), and mixtures thereof. Preferably, the complexing agent system comprises at least 10% by weight of MGDA and at least 10% by weight of citric acid. For purposes of the present invention, the term "acid" when referring to a complexing agent includes acids and their salts.

[0107] In a preferred embodiment, the composition comprises at least 15% by weight, more preferably 20% to 40% by weight, of MGDA, more preferably the trisodium salt of MGDA. Compositions with this high level of MGDA perform well in hard water and over long and / or high temperature cycles.

[0108] The complexing agent system of the present invention may further include citric acid.

[0109] 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 0.2 to about 15% by weight, more preferably 0.3 to % by weight.

[0110] The dispersant polymer is capable of suspending calcium or calcium carbonate in the automatic dishwashing process.

[0111] 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, make the following calcium binding capacity determination according to the instructions below.

[0112] Calcium binding capacity 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™ comb-type calcium 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 should be measured at 25°C. A stock solution of 1000 mg / g Ca is prepared by adding 3.67 g of CaCl2-2H2O to 1 L of deionized water, followed by dilution 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 of NaCl. 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. The test polymer is then added incrementally (at 0.01 g / L intervals) to the calcium working solution, with measurements taken after 5 minutes of stirring after each increment. 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.

[0113] The dispersant polymer preferably has a net negative charge when dissolved in an aqueous solution having a pH greater than 6.

[0114] 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 containing both sulfonated and carboxylated monomers.

[0115] Preferably, the dispersant polymer is a sulfonated derivative of a polycarboxylic acid and may contain two, three, four or more different monomer units. Preferred copolymers include:

[0116] At least one structural unit derived from a carboxylic acid monomer has the general formula (III):

[0117] [ka] wherein R1 to R3 are independently selected from hydrogen, methyl, a linear or branched saturated alkyl group having 2 to 12 carbon atoms, a linear or branched mono- or polyunsaturated alkenyl group having 2 to 12 carbon atoms, the aforementioned alkyl or alkenyl groups substituted with -NH2 or -OH, or -COOH, or COOR4, and wherein R4 is hydrogen, an alkali metal, or a linear or branched saturated or unsaturated alkyl or alkenyl group having 2 to 12 carbon atoms; 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, with acrylic and methacrylic acid being more preferred.

[0118] Optionally, one or more structural units derived from at least one nonionic monomer have the general formula (IV):

[0119] [ka] wherein R5 to R7 are independently selected from hydrogen, methyl, phenyl, or a hydroxyalkyl group containing 1 to 6 carbon atoms and can 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 a linear or branched, saturated alkyl group having 1 to 22 carbon atoms or an unsaturated, preferably aromatic, group having 6 to 22 carbon atoms.

[0120] 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- 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-methyl ... 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.

[0121] and at least one structural unit derived from at least one sulfonic acid monomer has the general formulas (V) and (VI):

[0122] [ka] wherein R7 is a group containing at least one sp2 bond, A is O, N, P, S, an amide, or an 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.

[0123] 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 foregoing acids or their water-soluble salts.

[0124] Preferably, the polymer contains the following concentrations of monomers: about 40 to about 90% by weight of the polymer, preferably about 60 to about 90% by weight of one or more carboxylic acid monomers; about 5 to about 50% by weight of the polymer, preferably about 10 to about 40% by weight of one or more sulfonic acid monomers; and optionally about 1 to about 30% by weight of the polymer, preferably about 2 to about 20% by weight of one or more nonionic monomers. Particularly preferred polymers contain about 70 to about 80% by weight of at least one carboxylic acid monomer and about 20 to about 30% by weight of the polymer of at least one sulfonic acid monomer.

[0125] 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 acid groups may be replaced by metal ions, preferably alkali metal ions, in particular sodium ions.

[0126] The carboxylic acid is preferably (meth)acrylic acid. The sulfonic acid monomer is preferably 2-acrylamido-2-propanesulfonic acid (AMPS).

[0127] 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.

[0128] Suitable dispersant polymers include low molecular weight anionic carboxylic acid polymers. They can 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 can also be a low molecular weight homopolymer of polyacrylate having an average molecular weight of 1,000 to 20,000, particularly 2,000 to 10,000, and particularly preferably 3,000 to 5,000.

[0129] 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, or a copolymer of acrylic acid and / or methacrylic acid and fumaric acid, having a molecular weight of less than 70,000, in the range of 2,000 to 80,000, more preferably 20,000 to 50,000, specifically 30,000 to 40,000 g / mol, with a ratio of (meth)acrylate to maleate or fumarate segments of 30:1 to 1:2.

[0130] The dispersant polymer may be a copolymer of acrylamide and acrylate having a molecular weight of 3,000 to 100,000, or alternatively 4,000 to 20,000, and an acrylamide content of less than 50%, or alternatively less than 20%, by weight of the dispersant polymer may 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.

[0131] Suitable dispersant polymers herein also include itaconic acid homopolymers and copolymers.

[0132] 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.

[0133] bleach-based The compositions of the present invention preferably comprise a bleaching system comprising a high concentration of bleach, 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.

[0134] 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.

[0135] 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 typically 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 a surface, or layer by layer.

[0136] Alkali metal percarbonates, especially sodium percarbonate, are preferred bleaching agents for use herein. The percarbonate is most preferably incorporated into the product in a coated form which provides in-product stability.

[0137] Potassium monopersulfate peroxide is another inorganic perhydrate salt useful herein.

[0138] 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.

[0139] Further typical organic bleaching agents include peroxyacids, specific examples being 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, and N,N-terephthaloyldi(6-aminopercaproic acid).

[0140] 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 that, under perhydrolysis conditions, give aliphatic peroxocarboxylic 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 specified number of carbon atoms 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 acid 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 present invention comprise from 0.01 to 5%, preferably from 0.2 to 2%, by weight of the composition of a bleach activator, preferably TAED.

[0141] 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.

[0142] Preferred bleaching catalysts for use herein include manganese triazacyclononane and related complexes; Co, Cu, Mn, and Fe bispyridylamines 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).

[0143] 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.

[0144] Inorganic Builders The compositions of the present invention preferably include an inorganic builder. Suitable inorganic builders are selected from the group consisting of carbonates, silicates, and mixtures thereof. Sodium carbonate is particularly preferred for use herein. Preferably, the compositions of the present invention include 5 to 60% by weight of the composition of sodium carbonate, more preferably 10 to 50% by weight, especially 15 to 45% by weight.

[0145] 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, particularly for the purpose of sheeting to avoid film formation and spot formation and improve gloss.It has been found that nonionic surfactant can also contribute to preventing redeposition of dirt.

[0146] Preferably, the compositions of the present invention comprise a nonionic surfactant or nonionic surfactant system, and more preferably, the nonionic surfactant or nonionic surfactant system has a phase inversion temperature of 40-70°C, preferably 45-65°C, when measured at a concentration of 1% in distilled water. "Nonionic surfactant system," as used herein, refers to 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.

[0147] 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 it 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.

[0148] The phase inversion temperature of a nonionic 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 predicted phase inversion temperature. The phase inversion temperature is determined visually at the first sign of turbidity.

[0149] Suitable nonionic surfactants include i) ethoxylated nonionic surfactants prepared by reacting a monohydroxyalkanol or alkylphenol 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; and ii) alcohol alkoxylated surfactants having 6 to 20 carbon atoms and at least one ethoxy group and one propoxy group. Mixtures of surfactants i) and ii) are preferred for use herein.

[0150] Other suitable nonionic surfactants are epoxy-capped poly(oxyalkylated) alcohols of 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.

[0151] Preferably, the surfactant of formula I has at least about 10 carbon atoms in the terminal epoxide unit [CHCH(OH)R]. In accordance with the present invention, a suitable surfactant of formula I is, for example, Olin Corporation's POLY-TERGENT® SLF-18B nonionic surfactant, as described in WO 94 / 22800, published October 13, 1994, by Olin Corporation.

[0152] enzyme Proteases The compositions of the invention can include a protease in addition to the amylase of the invention. A mixture of two or more enzymes can contribute to enhanced cleaning over a wider temperature, cycle duration, and / or substrate range, and can provide superior shine benefits, especially when used in conjunction with anti-redeposition agents and / or sulfonated polymers.

[0153] A suitable protease is a mutant subtilisin protease from Bacillus gibsonii having the amino acid substitutions X39E, X99R, X126A, X127E and X128G.

[0154] Another suitable protease is a subtilisin variant comprising three, four or five amino acid substitutions selected from the group consisting of S039E, S099R, S126A, D127E and F128G, and further comprising one or more additional substitutions selected from the group consisting of N74D, T114L, M122L, N198A, N198G, M211E, M211Q, N212Q and N242D, wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO:6.

[0155] Another suitable protease is (i) two or more amino acid substitutions selected from the group consisting of S039E, N74D, S099R, M211E, and N242D; and (ii) a subtilisin variant comprising one or more additional substitutions selected from the group consisting of T114L, M122L, S126A, F128G, N198A, N198G, M211Q, N212Q; The variant has at least 80% identity with the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.

[0156] Suitable proteases for use in combination with the amylases 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, plant, 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 and / or trypsin-type proteases. Examples of suitable neutral or alkaline proteases include: (a) Subtilisin (EC3.4.21.62), 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 Publication No. 10 Bacillus species, such as B. lentus (B.), as described in International Publication No. 2006022216(A1), International Publication No. 2015089447, International Publication No. 2015089441, International Publication No. 2016066756, International Publication No. 2016066757, International Publication No. 2016069557, International Publication No. 2016069563, International Publication No. 2016069569, International Publication No. 2016174234, International Publication No. 2017 / 089093, International Publication No. 2020 / 156419, and International Publication No. 2016 / 183509. those from the genus Bacillus, such as B. lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, B. pumilus, B. gibsonii, and B. akibaii; Specifically, subtilisins include the mutations S9R, A15T, V66A, A188P, V199I, N212D, Q239R, N255D, X9E, X200L, X256E, X9R, X19L, X60D (Savinase numbering system); variants comprising an amino acid substitution at at least one of positions 9, 130, 133, 144, 224, 252, 271 (BPN' numbering system) derived from B. pumilus, such as those described in DE 102006022224(A1), WO 2020 / 221578, WO 2020 / 221579, and WO 2020 / 221580. (b) trypsin- 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.

[0157] Additional particularly preferred proteases 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%, especially 100% identity to SEQ ID NO: 7, and which variants comprise substitutions at one or more, or two or more, or three or more of the following positions relative to SEQ ID NO: 7: 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

[0158] Preferred proteases include the following mutations: 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.

[0159] Other suitable proteases include (a) a protease having at least 80% sequence identity to the sequence of SEQ ID NO: 6 and comprising three or more substitutions selected from A37T, S39E, I43V, A47V, P54T, T56Y, I80V, N85S, E87D, S99R, T114Q, M122L, S126A, D127E, F128G, N198A, M211Q, N212Q and N242D, wherein the numbering is according to SEQ ID NO: 6; (b) a protease having at least 80% sequence identity to the sequence of SEQ ID NO: 8 and containing one or more substitutions selected from Q12L, I21V, I43V, M122L, D127P, N154S, T156A, G160S, N177V, M211N, M211S, M211L, P212D, P212H, A222S, V228I and T247N, wherein the numbering is according to SEQ ID NO: 8; and (c) a protease having at least 80% sequence identity with the sequence of SEQ ID NO:9 and comprising three or more substitutions selected from S9R, A15T, G59E, V66A, H118N, A188P, V199I, Q200E, N212D, Q239R, N255D, wherein the numbering is according to SEQ ID NO:9.

[0160] Suitable commercially available additional 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 Figure 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) manufactured by Kao; and Lavergy®, Lavergy® Pro, and Lavergy® C Bright manufactured by BASF.

[0161] 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 Ultra®, Blaze Evity® and Blaze Pro®, BLAP and BLAP variants.

[0162] Preferred concentrations of protease in the products of the present invention include about 0.05 to about 10, more preferably about 0.5 to about 7, and especially about 1 to about 6 mg of active protease per gram of composition.

[0163] Other amylases Preferably, the compositions of the present invention may contain amylase. Suitable alpha-amylases include those of bacterial or fungal origin, including chemically or genetically modified variants. Preferred alkaline alpha-amylases are derived from Bacillus species, such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis, or other Bacillus species, such as Bacillus species NCBI 12289, NCBI 12512, NCBI 12513, DSM 9375 (U.S. Pat. No. 7,153,818), DSM 12368, DSM Z No. 12649, KSM AP1378 (WO 97 / 00324), KSM K36, or KSM K38 (EP 1,022,334). Preferred amylases include:

[0164] Other amylases include: (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: having one or more substitutions at positions 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 * Variants that also contain deletions. (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 with deletions at positions 183 and 184, and 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 to the wild-type enzyme from Bacillus sp. 707 (SEQ ID NO: 7 in U.S. Pat. No. 6,093,562), particularly those containing one or more of the following mutations: M202, M208, S255, R172, and / or M261. Preferably, the amylase contains one or more of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N, and / or R172Q. Particularly preferred are those containing the M202L or M202T mutation. (d) A variant described in WO 09 / 149130, preferably SEQ ID NO: 1 or SEQ ID NO: 2 of WO 09 / 149130, which shows at least 90% identity to the wild-type enzyme from Geobacillus Stearophermophilus or a truncated version thereof. (e) Variants exhibiting at least 89% identity with SEQ ID NO: 1 of WO2016091688, in particular those 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 showing at least 85% identity with 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 with the mature amino acid sequence of Alicyclobacillus sp. amylase (SEQ ID NO: 8 in WO 2016180748).

[0165] Preferably, the amylase is a genetically engineered enzyme in which one or more amino acids susceptible to bleaching oxidation are replaced with amino acids less susceptible to oxidation. Specifically, methionine residues are preferably replaced with any other amino acid. Specifically, the methionine most susceptible to oxidation is preferably replaced. Preferably, the methionine at position 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 with threonine or leucine, preferably leucine.

[0166] 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.

[0167] 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.

[0168] Preferably, the protease and / or amylase of the compositions of the present 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.

[0169] Protease Stabilizers Peptide aldehydes may be used as protease stabilizers in detergent formulations, as previously described (WO 199813458, WO 2011036153, U.S. Patent Application Publication No. 20140228274). Examples of peptide aldehyde stabilizers are peptide aldehydes, ketones, or halomethyl ketones, which may be "N-capped," e.g., with a ureido, carbamate, or urea moiety, or "doubly N-capped," e.g., with a carbonyl, ureido, oxamide, thioureido, dithiooxamide, or thiooxamide moiety (EP 2358857(B1)). The molar ratio of these inhibitors to protease may be 0.1:1 to 100:1, e.g., 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 (B2)). The molar ratio of these stabilizers to protease 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.

[0170] Crystal Growth Inhibitors Crystal growth inhibitors are materials that can bind to calcium carbonate crystals and prevent further growth of species such as aragonite and calcite.

[0171] Examples of effective crystal growth inhibitors include phosphonates, polyphosphonates, inulin derivatives, polyitaconic acid homopolymers, and cyclic polycarboxylates.

[0172] 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 form and the protonated carboxylic acid form.

[0173] The cyclic carboxylate contains 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, bicycloheptane or bicyclooctane, and / or tetrahydrofuran. One preferred crystal growth inhibitor is cyclopentanetetracarboxylate.

[0174] 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.

[0175] Preferably, the two carboxylate groups on the same side of the ring are directly adjacent or in the "ortho" position.

[0176] Preferred crystal growth inhibitors include HEDP, tricarballylic acid, tetrahydrofurantetracarboxylic acid (THFTCA), and cyclopentanetetracarboxylic acid (CPTCA). THFTCA preferably has a 2c,3t,4t,5c configuration, and CPTCA has a cis,cis,cis,cis configuration. A particularly preferred crystal growth inhibitor for use herein is HEDP.

[0177] 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.

[0178] The crystal growth inhibitor is preferably present in an amount of from about 0.01 to about 10% by weight of the composition, especially from about 0.02 to about 5% by weight, and especially from 0.05 to 3% by weight.

[0179] 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 to 5% by weight of the product, more preferably 0.2 to 4% by weight, especially 0.3 to 3% by weight of the metal care agent, and preferably the metal care agent is benzotriazole (BTA).

[0180] Glass care products Glass care agents protect the appearance of glassware during the dishwashing process. Preferably, the compositions of the present invention contain 0.1 to 5% by weight of the composition, more preferably 0.2 to 4% by weight, and especially 0.3 to 3% by weight of a metal care agent. Preferably, the glass care agent is a zinc-containing material, especially zincite. Another suitable glass care agent is polyethyleneimine (PEI). A particularly preferred PEI is Lupasol® FG, supplied by BASF.

[0181] 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% wt / vol aqueous solution in distilled water at 20°C.

[0182] 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 a pH of 9.5 when measured in NaOH with 100 grams of product at 20°C.

[0183] Washing conditions There are a variety of wash conditions, including various detergent formulations, wash water volumes, wash water temperatures, and wash time lengths, to which one or more amylases described herein may be exposed. Low-concentration detergent systems are intended for wash water containing less than about 800 ppm of detergent ingredients. Medium-concentration detergent systems are intended for washes containing from about 800 ppm to about 2000 ppm of detergent ingredients. High-concentration detergent systems are intended for wash water containing more than about 2000 ppm of detergent ingredients. 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 ranges of about 15°C to about 35°C or 10°C to 40°C.

[0184] Water hardness varies depending on the geography. Hardness is determined by the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) is a measure of the amount of Ca 2+ / Mg 2+ Hardness is expressed in grains per gallon (gpg) of a mixture of minerals. Most water in the United States is hard, but hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has hardness minerals between 60 and 181 ppm (you can convert ppm to grains per U.S. gallon by dividing ppm by 17.1).

[0185] [Table 3]

[0186] Embodiments of the present invention The following are embodiments of the present invention: 1. A home care composition comprising a surfactant and an amylase, wherein the amylase is a recombinant, non-naturally occurring variant of a parent α-amylase, and the variant α-amylase has at least 80% identity, preferably at least 85% identity, preferably at least 86% identity, preferably at least 87% identity, preferably at least 88% identity, preferably at least 89% identity, preferably at least 90% identity, preferably at least 95% identity, preferably at least 96% identity, preferably at least 97% identity, preferably at least 98% identity, or preferably at least 99% identity to SEQ ID NO:5, and has an amino acid substitution at position 51 and / or 125 relative to SEQ ID NO:5. 2. The composition of embodiment 1, wherein the amylase comprises the amino acid substitutions T51V and / or S125R with respect to SEQ ID NO:5. 3. The composition of embodiment 1 or 2, wherein the amylase comprises an amino acid substitution at positions 172, 227 and / or 231 relative to SEQ ID NO:5. 4. The composition of embodiment 3, wherein the amylase comprises amino acid substitutions N172Q, N227R and / or F231L with respect to SEQ ID NO:5. 5. The amylase has an amino acid substitution with respect to SEQ ID NO:5: (a) T51V+S125R+F231L; (b) T51V + S125R + N172Q + N227R; or (c)N29Q+T51V+S125R+N227R+S253L+G272E+K319R+S418A, may have 6. The composition of any one of embodiments 1 to 5, further comprising a variant subtilisin protease from Bacillus gibsonii having the amino acid substitutions X39E, X99R, X126A, X127E and X128G. 7. The composition of any one of claims 1 to 6, wherein the composition is an automatic dishwashing composition. 8. The composition according to any one of claims 1 to 7, wherein the composition comprises a bleaching system. 9. The composition of any one of embodiments 1-8, 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. 10. The composition comprises 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, metalloprotease, 10. The composition of any one of embodiments 1-9, comprising one or more other enzymes selected from enzymes selected from: enzymes, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, PETases, phenoloxidases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonases, tannases, transglutaminases, xylan acetyl esterases, xylanases, and xylosidases; and combinations thereof. 11. The composition of embodiment 10, wherein the one or more enzymes comprise a protease, and the protease is a subtilisin variant comprising three, four, or five amino acid substitutions selected from the group consisting of S039E, S099R, S126A, D127E, and F128G, and further comprising one or more additional substitutions selected from the group consisting of N74D, T114L, M122L, N198A, N198G, M211E, M211Q, N212Q, and N242D, and wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO:6. 12.1 or more enzymes include proteases, and the proteases are (i) two or more amino acid substitutions selected from the group consisting of S039E, N74D, S099R, M211E, and N242D; and (ii) a subtilisin variant comprising one or more additional substitutions selected from the group consisting of T114L, M122L, S126A, F128G, N198A, N198G, M211Q, N212Q; The variant has at least 80% identity with the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7. 13.1 or more enzymes include proteases, and the proteases are (a) a protease having at least 80% sequence identity to the sequence of SEQ ID NO: 6 and comprising three or more substitutions selected from A37T, S39E, I43V, A47V, P54T, T56Y, I80V, N85S, E87D, S99R, T114Q, M122L, S126A, D127E, F128G, N198A, M211Q, N212Q and N242D, wherein the numbering is according to SEQ ID NO: 6; (b) a protease having at least 80% sequence identity to the sequence of SEQ ID NO: 8 and containing one or more substitutions selected from Q12L, I21V, I43V, M122L, D127P, N154S, T156A, G160S, N177V, M211N, M211S, M211L, P212D, P212H, A222S, V228I and T247N, wherein the numbering is according to SEQ ID NO: 8; and (c) a protease having at least 80% sequence identity to the sequence of SEQ ID NO:9 and comprising three or more substitutions selected from S9R, A15T, G59E, V66A, H118N, A188P, V199I, Q200E, N212D, Q239R, N255D, wherein the numbering is according to SEQ ID NO:9. 14. A cleaning method comprising contacting a surface or item in need of cleaning with an effective amount of the composition of any one of embodiments 1-13, optionally further comprising rinsing the surface or item after contacting the surface or item with the variant or enzyme composition. [Example]

[0187] Example 1. AA2560 α-amylase variant Protein expression, purification, and quantification: AA2560 α-amylase combinatorial variants based on the AA2560 α-amylase variant described in WO 2021 / 080948 (SEQ ID NO: 5 herein) were generated as synthetic genes and introduced into appropriate Bacillus licheniformis cells using standard procedures. All mutations were confirmed by DNA sequencing. Cells were grown for 72 hours in a medium suitable for protein expression and secretion in the B. licheniformis host. Secreted proteins were harvested by centrifugation. Purification was achieved by hydrophobic interaction chromatography using Phenyl Sepharose 6 Fast Flow Resin (GE Healthcare). The purified proteins were stabilized in a standard formulation buffer containing HEPES as a pH 8 buffer, calcium chloride, and propylene glycol. Protein concentration was determined by a combination of amino acid analysis, high-performance liquid chromatography (HPLC), and absorbance at 280 nm.

[0188] Enzyme Performance Assay: α-Amylase activity was determined by removing dyed starch stains from white melamine tiles in a detergent background. The cleaning activity of α-amylase was determined using tiles of mixed corn / rice colored starch and mixed corn / rice starch with food coloring, purchased from the Center for Test Materials (catalog no. DM277). The tiles were fixed into a 96-well plate containing amylase solutions diluted to the working range in aqueous buffer and added to a pre-made detergent solution of WFKB detergent (WFK Testgewebe GmbH, Bruggen, Germany), resulting in a total volume of 300 μL. The pre-imaged melamine tile with the colored starch stain was then fixed on top of the 96-well plate, such that agitation of the assembly resulted in splashing of the enzyme-containing detergent onto the surface of the starch stain. The cleaning reaction was carried out at 50°C for 15 minutes with shaking at 250 rpm. After the cleaning reaction, the melamine tiles were briefly rinsed with water, dried, and re-imaged. The activity of α-amylase is calculated as the difference in RGB (color) values ​​of the images before and after washing. The whiter the image after washing, the better the enzyme activity. The performance index (PI) is calculated as follows:

[0189]

number

[0190] Figure of merit for combinatorial mutants relative to ΔRG mutant: The cleaning performance of the variants in terms of performance index relative to the variant of SEQ ID NO: 5 is listed in Table 3.

[0191] [Table 4]

[0192] All variants in Table 3 perform better than the variant in SEQ ID NO:5.

[0193] 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. A home care composition comprising a surfactant and an amylase, wherein the amylase is a recombinant, non-naturally occurring variant of a parent α-amylase, the variant α-amylase having at least 90% identity to SEQ ID NO:5, wherein the amylase comprises the amino acid substitutions T51V and S125R relative to SEQ ID NO:5, and is a maltopentaose / maltohexaose forming amylase.

2. The composition described in claim 1, wherein the mutant α-amylase has at least 95% identity with sequence number 5.

3. The composition described in claim 1, wherein the mutant α-amylase has at least 99% but less than 100% identity to SEQ ID NO:

5.

4. 2. The composition of claim 1, wherein the amylase comprises amino acid substitutions N172Q, N227R and / or F231L relative to SEQ ID NO:

5.

5. The amylase may comprise the amino acid substitution: Regarding SEQ ID NO: 5 (a) T51V+S125R+F231L; (b) T51V+S125R+N172Q+N227R; or (c) N29Q+T51V+S125R+N227R+S253L+G272E+K319R+S418A, The composition of claim 1 comprising:

6. The composition of claim 1 , wherein the composition is an automatic dishwashing composition.

7. The composition of claim 1 , wherein the composition comprises a bleaching system.

8. 10. The composition of claim 1, 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.

9. 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 mannanase, a metalloproteinase, a hydroxybenzoate ...

10. The composition of claim 1, comprising one or more other enzymes selected from proteases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, PETases, phenoloxidases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonases, tannases, transglutaminases, xylan acetyl esterases, xylanases, and xylosidases; and combinations thereof.

10. 10. A method of cleaning comprising contacting a surface or item in need of cleaning with an effective amount of a composition according to any one of claims 1 to 9, optionally further comprising rinsing the surface or item after contacting the surface or item with the variant or enzyme composition.

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