α-Amylase combination mutants

Abstract

Disclosed are compositions and methods related to variant α-amylases that are useful, for example, for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing and other applications, for textile processing (e.g., desizing), in animal feed to improve digestibility, and for baking and brewing. [Selected Figure] Figure 1

Description

[Technical field]

[0001] Priority This application claims the benefit of U.S. Provisional Patent Application No. 62 / 479,726, filed March 31, 2017, which is hereby incorporated by reference in its entirety.

[0002] Disclosed are compositions and methods relating to variant α-amylases containing multiple combinable mutations that are useful, for example, for starch liquefaction and saccharification, cleaning starchy stains, fabric desizing, baking and brewing. [Background technology]

[0003] 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.

[0004] Starch-derived sugars in the form of concentrated dextrose syrup are currently produced by an enzyme-catalyzed process that involves (1) gelatinization and liquefaction (or viscosity reduction) of solid starch to dextrins with an average degree of polymerization of about 7-10 using α-amylase, and (2) saccharification of the resulting liquefied starch (i.e., starch hydrolysate) with amyloglucosidase (also called glucoamylase or GA). The resulting syrup has a high glucose content. Much of the commercially produced glucose syrup is subsequently enzymatically isomerized to a dextrose / fructose mixture known as isosyrup. The resulting syrup can also be fermented using microorganisms, such as yeast, to produce commercial products that contain, for example, ethanol, citric acid, lactic acid, succinic acid, itaconic acid, monosodium glutamate, gluconate, lysine, other organic acids, other amino acids, and other biochemicals. Fermentation and saccharification can be carried out simultaneously (i.e., SSF processes) to achieve greater economy and efficiency.

[0005] Alpha-amylases hydrolyze starch, glycogen and related polysaccharides by randomly cleaving internal alpha-1,4-glucosidic bonds. Alpha-amylases, particularly from the Bacillus genus, have been used for a wide variety of purposes, including starch liquefaction and saccharification, textile desizing, starch modification in the pulp and paper industry, brewing, baking, and production of syrups for the food industry, 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 and laundry washing.

[0006] Many publications have described mutations in α-amylases. However, not all mutations produce the same effect in different molecules, and not all mutations can be combined. Furthermore, many mutations produce molecules with certain desirable properties at the expense of other properties. There is a need for robust engineered α-amylase molecules. Summary of the Invention [Means for solving the problem]

[0007] The present compositions and methods relate to variant amylase polypeptides and methods of their use. Aspects and embodiments of the present compositions and methods are summarized in the individually numbered paragraphs below. 1. In one embodiment, a recombinant variant of a parent α-amylase comprising: (a) a deletion of at least one amino acid residue corresponding to R181, G182, T183 and G184, using SEQ ID NO: 1, and optionally mutations at amino acid residues corresponding to T183, E190, M202, Q172, A186 and / or I324, wherein the variant α-amylase or parent α-amylase has at least 91%, optionally 92%, optionally 93%, optionally 94%, optionally 95%, optionally 96%, optionally 97%, optionally 98% or optionally 99% amino acid sequence identity to SEQ ID NO: 1, used for numbering purposes, and wherein the variant has improved cleaning performance in automatic dishwashing compared to a reference α-amylase that differs from the parent α-amylase or variant α-amylase only by the absence of the mutations, or (b) A recombinant variant of a parent α-amylase is provided, which comprises one or several amino acid substitutions, deletions or additions in the amino acid sequence of the variant in (a) and has the activity of the variant in (a). 2. In some embodiments, the variant α-amylase of paragraph 1 comprises a deletion of amino acid residues corresponding to R181 and G182, or T183 and G184, using SEQ ID NO:1 for numbering. 3. In some embodiments, the variant α-amylase of any of paragraphs 1-2 has a variant α-amylase having a nucleotide sequence similar to that of any of the following positions: T183 and E190, T183 and M202, E190 and M202, T183, E190 and M202, Q172 and A186, A186 and I324, Q172 and I324, Q172, A186 and I324, Q172, A186 and M202, A186, I324 and M202, Q172, I324 and M202 or Q172, A186, I324 and M202 has a mutation in 4. In some embodiments, the mutant α-amylase of any of paragraphs 1-3 comprises one or more of the following mutations: T183D and E190P, T183D and M202L, T183D, E190P and M202L, Q172R and A186G, A186G and I324M, Q172R and I324M, Q172R, A186G and I324M, Q172R, A186G and M202L, A186G, I324M and M202L, Q172R, I324M and M202L or It has Q172R, A186G, I324M and M202L. 5. In some embodiments of the mutant α-amylase of any of paragraphs 1-4, the mutant is encoded by a polynucleotide that hybridizes under stringent conditions to the polynucleotide of SEQ ID NO:2, or its complement. 6. In yet another aspect, there is provided a polynucleotide encoding the mutant amylase of any of paragraphs 1 to 5, an expression vector comprising the polynucleotide, or an expression host comprising the polynucleotide or expression vector. 7. In yet another aspect, there is provided a composition for liquefying starch comprising the variant amylase of any of paragraphs 1-5. 8. In yet another aspect, there is provided a detergent composition comprising the variant amylase of any of paragraphs 1-5. 9. In yet another aspect, there is provided a method for converting starch to oligosaccharides, comprising contacting starch with an effective amount of a variant amylase of any of paragraphs 1-5. 10. In yet another aspect, there is provided a method for removing starchy stains or soils from a surface comprising contacting the surface with an effective amount of a variant amylase of any of paragraphs 1-4 and enabling the polypeptide to hydrolyze starch components present in the starchy stains to produce smaller starch-derived molecules that are soluble in an aqueous composition, thereby removing the starchy stains from the surface.

[0008] These and other aspects and embodiments of the present compositions and methods will become apparent from the description and drawings herein. [Brief description of the drawings]

[0009] [Figure 1] 1 is a graph showing the starch cleaning performance of enzymes in CASCADE® PLATINUM™ dishwasher detergent formulations containing deactivated enzymes.

[0010] [Diagram 2] 1 is a graph showing the starch cleaning performance of enzymes in CASCADE® PLATINUM™ dishwasher detergent formulations containing deactivated enzymes.

[0011] [Diagram 3] 1 is a graph showing the starch cleaning performance of enzymes in CASCADE® PLATINUM™ dishwasher detergent formulations containing deactivated enzymes.

[0012] [Figure 4] 1 is a graph showing the starch cleaning performance of enzymes in FINISH® QUANTUM™ dishwasher detergent formulations containing deactivated enzymes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Compositions and methods relating to variant α-amylase enzymes are described. Typical uses of variant amylase enzymes are for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing and other applications, for the textile processing industry (e.g., desizing), for improving digestibility in animal feed, and for baking and brewing. These and other aspects of the compositions and methods are described in detail below.

[0014] Before describing the various aspects and embodiments of the present compositions and methods, the following definitions and abbreviations are provided.

[0015] 1. Definitions and Abbreviations In accordance with this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "enzyme" includes a plurality of such enzymes, and reference to a "dosage" includes one or more doses and equivalents thereof known to those skilled in the art.

[0016] This specification is organized into sections for ease of reading. However, the reader will understand that statements made in one section may be applicable to other sections. Thus, the headings used in the different sections of this disclosure should not be construed as limiting.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following terms are provided below.

[0018] 1.1. Abbreviations and Acronyms The following abbreviations / acronyms have the following meanings unless otherwise specified: DNA deoxyribonucleic acid EC Enzyme Committee FH French hardness GA glucoamylase GH General hardness HDL High Density Liquid Detergent HDD Powerful Powder Detergent HSG High Foam Granular Detergent HFCS High Fructose Corn Syrup IRS Insoluble residual starch kDa kilodalton MW molecular weight MWU Modified Wohlgemuth Unit; 1.6×10 -5 mg / MWU = activity units NCBI National Center for Biotechnology Information PI figure of merit ppm parts per million, e.g., μg protein per gram of dry solids RCF Relative centrifugal force / centripetal force (i.e., x gravity) RNA Ribonucleic acid sp. species Tm melting temperature w / v weight / volume w / w weight / weight v / v volume / volume wt% weight% ℃ Celsius temperature H2O Water dH2O or DI deionized water dIH2O Deionized water, Milli-Q filtered g or gm grams μg microgram mg milligram kg Kilogram μL and μl microliter mL and ml milliliters mm millimeters μm micrometer M mole mM millimolar μM micromolar U Units sec seconds min(s) minutes hr(s) time ETOH Ethanol N Regulation MWCO Molecular Weight Cutoff CAZy carbohydrate-related enzyme database

[0019] 1.2.Definition of Terms The term "amylase" or "amylolytic enzyme" refers specifically to an enzyme capable of catalyzing the degradation of starch. α-amylase is a hydrolase that cleaves α-D-(1→4) O-glycosidic bonds in starch. In general, α-amylase (EC 3.2.1.1; α-D-(1→4)-glucan glucanohydrolase) is defined as an endo-acting enzyme that cleaves α-D-(1→4) O-glycosidic bonds in starch molecules in a random manner to produce polysaccharides containing three or more (1-4)-α-linked D-glucose units. In contrast, some product-specific amylases, such as exo-acting amylolytic enzymes, e.g., 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 concentrated syrups of specific length malto-oligosaccharides or specific malto-oligosaccharides.

[0020] The term "starch" means a compound of the formula (CH 10 O5) X"starch" refers to any material composed of complex polysaccharide carbohydrates of plants composed of amylose and amylopectin having the formula: (wherein X can be any number). The term includes materials obtained from plant-based materials such as cereals, cereals, grasses, tubers and roots, and more particularly 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, i.e. uncooked starch, e.g., starch that has not been gelatinized.

[0021] The terms "wild-type," "parent," or "reference" in reference 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 "wild-type," "parent," or "reference" in reference to a polynucleotide refer to a naturally occurring polynucleotide that does not contain an artificial nucleoside change. However, it should be noted that a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to naturally occurring polynucleotides, but encompasses any polynucleotide that encodes a wild-type, parent, or reference polypeptide.

[0022] 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., cleaved from the immature form of the polypeptide during or after expression of the polypeptide.

[0023] The term "variant" with respect to a polypeptide refers to a polypeptide that differs from a particular 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 particular wild-type, parent, or reference polynucleotide. The identity of the wild-type, parent, or reference polypeptide or polynucleotide will be clear from the context.

[0024] In the case of the present α-amylases, "activity" refers to α-amylase activity, which can be measured as described herein.

[0025] The term "performance advantage" refers to an improvement in a desirable property of a molecule. Typical performance advantages 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 heat resistance, increased detergent stability, increased storage stability, increased solubility, altered pH profile, decreased calcium dependency, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability and increased expression. In some cases, the performance advantage is realized at significantly lower temperatures. In some cases, the performance advantage is realized at significantly higher temperatures.

[0026] The terms "protease" and "proteinase" refer to enzyme proteins capable of performing "proteolysis" or "proteolytic cleavage," which means the hydrolysis of the peptide bonds that link amino acids together within the peptide or polypeptide chains that form proteins. This activity of proteases as protein-digesting enzymes is called "proteolytic activity."

[0027] The term "serine protease" refers to enzymes that cleave peptide bonds in proteins in which serine functions as the nucleophilic amino acid at the enzyme active site. Serine proteases are classified into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like. It is the serine proteases, particularly the subtilisins, that are most commonly used in laundry and dishwashing detergents.

[0028] A "combination variant" is a variant that contains two or more mutations, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, deletions and / or insertions.

[0029] A "combinable mutation" is a mutation at any amino acid position that can be used to create a combinatorial variant. A combinatorial mutation improves at least one desired property of a molecule (in this case, an amylase) without significantly decreasing either expression, activity, or stability.

[0030] The term "recombinant" when used with respect to a subject cell, nucleic acid, protein, or vector indicates that the subject has been modified 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 a native sequence by one or more nucleotides and / or is operably linked to a heterologous sequence, such as a heterologous promoter in an expression vector. A recombinant protein may differ from a native sequence by one or more amino acids and / or is fused to a heterologous sequence. A vector containing a nucleic acid encoding an amylase is a recombinant vector.

[0031] The terms "recovered," "isolated," and "separated" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other particular substance or component that has been removed from at least one other substance or component with which it is naturally associated as found in nature. Such "isolated" polypeptides include, but are not limited to, cell culture broth containing secreted polypeptides expressed in a heterologous host cell.

[0032] The term "purified" refers to a material (e.g., an isolated polypeptide or polynucleotide) that is in a substantially 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.

[0033] The term "enriched" refers to a material (eg, 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.

[0034] The terms "thermostable" and "thermostability" in reference to enzymes refer to the ability of an enzyme to retain activity after exposure to high temperature. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (t1 / 2), given in minutes, hours or days during which half of the enzyme activity is lost under defined conditions. The half-life can be calculated by measuring the residual α-amylase activity after exposure to high temperature (i.e., challenge with high temperature).

[0035] "pH range" in reference to an enzyme refers to the range of pH values ​​over which the enzyme exhibits catalytic activity.

[0036] The terms "pH stable" and "pH stability" in relation to an enzyme refer to the ability of the enzyme to retain activity over a wide pH range for a defined period of time (eg, 15 minutes, 30 minutes, 1 hour).

[0037] The term "amino acid sequence" is synonymous with, and is used interchangeably with, the terms "polypeptide," "protein," and "peptide." When such amino acid sequences exhibit activity, they can be referred to as "enzymes." Conventional one-letter or three-letter codes are used for amino acid residues, and amino acid sequences are represented in the standard amino to carboxy terminal direction (i.e., N→C).

[0038] The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single-stranded or double-stranded and can contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to code for a particular amino acid, and the present compositions and methods encompass nucleotide sequences that code for a particular amino acid sequence. Unless otherwise specified, nucleic acid sequences are presented in the 5'- to 3'-direction.

[0039] "Hybridization" refers to the process by which one strand of nucleic acid forms a duplex, i.e., base pairs, with a complementary strand, as occurs during blot hybridization and PCR techniques. Stringent hybridization conditions are exemplified by hybridization at the following conditions: 65°C and 0.1xSSC, where 1xSSC = 0.15M NaCl, 0.015M NaCitrate, pH 7.0. A hybridized double-stranded nucleic acid is characterized by a melting temperature (Tm) at which one-half of the hybridized nucleic acid does not pair with the complementary strand. Mismatched nucleotides within the duplex decrease the Tm. A nucleic acid encoding a mutant α-amylase may have a Tm that is 1°C to 3°C or more lower than a duplex formed between the nucleotides of SEQ ID NO:2 and its identical complement.

[0040] A "synthetic" molecule is produced not by an organism, but rather by in vitro chemical or enzymatic synthesis.

[0041] The terms "transformed," "stably transformed," and "transgenic" as used with respect to cells mean that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.

[0042] In reference to inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection," "transformation," or "transduction," as known in the art.

[0043] 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. Typical 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.

[0044] The term "heterologous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

[0045] The term "endogenous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in a host cell.

[0046] The term "expression" refers to the process by which a polypeptide is produced based on a nucleic acid sequence. This process includes both transcription and translation.

[0047] "Selective marker" or "selectable marker" refers to a gene that can be expressed in a host to facilitate the selection of host cells that carry the gene. Examples of selectable markers include, but are not limited to, antibiotics (e.g., hygromycin, bleomycin, or chloramphenicol) and / or genes that confer a metabolic advantage, e.g., a nutritional advantage, to the host cell.

[0048] "Vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, etc.

[0049] "Expression vector" means a DNA construct containing a DNA sequence encoding a polypeptide of interest, the coding sequence being operably linked to suitable control sequences capable of effecting the expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, an enhancer, and sequences which control the termination of transcription and translation.

[0050] The term "operably linked" refers to a relationship (including, but not limited to, juxtaposition) that permits particular components to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence.

[0051] 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 off during the secretion process.

[0052] "Biologically active" refers to a sequence that has a particular biological activity, such as, for example, enzymatic activity.

[0053] 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 of protein.

[0054] As used herein, "water hardness" is a measure of the minerals (eg, calcium and magnesium) present in water.

[0055] A "swatch" is a piece of material, such as a textile, having a stain applied thereto. The material may be, for example, a textile made from cotton, polyester or a mixture of natural and synthetic fibers. The swatch may also be a piece of paper, such as filter paper or nitrocellulose, or a piece of hard material, such as ceramic, metal or glass. For amylase, the stain is starch-based, but may also include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigment, oil or a mixture of these compounds.

[0056] A "small swatch" is a piece of swatch cut using a single hole punch or a custom-made 96-hole punching device where the pattern of the multi-hole punch fits into a standard 96-well microtiter plate or the piece is otherwise removed from the swatch. The swatch may be a swatch of fabric, paper, metal, or other suitable material. The small swatch may have a stain applied to it either before or after it is placed into the well of a 24-well, 48-well, or 96-well microtiter plate. The small swatch may also be created by applying a stain to a small piece of material. For example, the small swatch may be a stained piece of fabric 5 / 8" or 0.25" in diameter. The custom-made punching machine is designed in a way to simultaneously deliver 96 pieces of swatches to all wells of a 96-well plate. This device allows for the delivery of more than one swatch per well by simple multiple loading into the same 96-well plate. The multi-hole punch can be envisioned to simultaneously deliver swatches to any format plate, including but not limited to 24-well, 48-well or 96-well plates. In another envisioned method, the soil test platform can be a bead made of metal, plastic, glass, ceramic or other suitable material that is coated with a solid substrate. One or more coated beads are then placed into the wells of a 96-well, 48-well or 24-well plate or larger format containing suitable buffers and enzymes.

[0057] "Cultured cellular 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 that is grown in culture for the purpose of producing amylase.

[0058] "Percent sequence identity" means that a particular sequence has at least a specified percentage of amino acid residues that are identical to the amino acid residues in a particular reference sequence when aligned using 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: Gap Start Penalty: 10.0 Gap extension penalty: 0.05 Protein Weight Matrix: BLOSUM Series DNA Weight Matrix: IUB Delay divergence array (%): 40 Gap Separation Distance: 8 DNA transition weight: 0.50 List of hydrophilic residues: GPSNDQEKR Use negative matrix:OFF Toggle residue-specific penalty: ON Toggle Hydrophilic Penalty: ON Toggle End Gap Separation Penalty:OFF

[0059] Deletions are counted as non-identical residues compared to the reference sequence.

[0060] A "fused" polypeptide sequence is one in which two subject polypeptide sequences are connected, ie, operably linked, by a peptide bond between them.

[0061] The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina, in particular the Pezizomycotina species.

[0062] The term "dry solids content" (ds) refers to the total solids of the slurry on a dry weight % basis. The term "slurry" refers to an aqueous mixture containing insoluble solids.

[0063] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process in the production of biochemicals in which a microbial organism, e.g., an ethanol-producing microorganism, and at least one enzyme, e.g., 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 biological materials in the same reaction vessel.

[0064] "Ethanologenic microorganism" refers to a microorganism with the ability to convert sugars or oligosaccharides into ethanol.

[0065] The term "fermented beverage" refers to any beverage produced by a process that includes a fermentation process, such as microbial fermentation, such as bacterial and / or fungal fermentation. "Beer" is an example of such a fermented beverage, and the term "beer" is intended to include any fermented wort produced by fermentation / brewing of starch-containing plant material. Beer is often produced exclusively from malt or additives or any combination of malt and additives.

[0066] The term "malt" refers to any malted grain, for example malted barley or wheat.

[0067] The term "additive" refers to any starch and / or sugar containing vegetable matter that is not malted, such as, for example, barley malt or wheat malt. Examples of additives include common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehulled barley, wheat, wheat starch, roasted grains, grain flakes, rye, oats, potato, tapioca, cassava, and syrups, such as, for example, corn syrup, cane syrup, invert sugar, barley and / or wheat syrup.

[0068] The term "mash" refers to an aqueous slurry of vegetable matter, such as milling grains, e.g., cracked barley malt, any starch and / or sugar containing other additives, or combinations thereof, admixed with water, which is subsequently separated into mash and spent grains.

[0069] The term "wort" refers to the unfermented liquor released after extraction of ground grains during mashing.

[0070] The term "about" refers to ±15% of a reference value.

[0071] 2. α-Amylase variants One embodiment of the present compositions and methods is a mutant α-amylase enzyme that contains a combination of mutations that improve their performance in industrial applications. The combined mutant is based on an α-amylase from Bacillus sp. and is referred to herein as "BspAmy24." The amino acid sequence of the mature form of the BspAmy24 α-amylase polypeptide is set forth below as SEQ ID NO:1. [ka]

[0072] Using SEQ ID NO:1 as a starting point, numerous combinatorial variants were generated and tested with an emphasis on identifying variants with high levels of cleaning performance under North American automatic dishwashing (ADW) conditions. Several variants were identified that had excellent cleaning performance.

[0073] Some, but not all, of the best performing mutant BspAmy24 α-amylases contained deletions within the X1G / S1X2G2 motif adjacent to the calcium binding loop, corresponding to R181, G182, T183, and G184, using SEQ ID NO:1 (underlined above) for numbering. Pairwise deletions of amino acid residues corresponding to R181 and G182 or T183 and G184 are generally equally effective in improving stability and reducing calcium dependence, due to the nature of the X1G / S1X2G2 motif. Deletion of amino acid residues corresponding to R181 and G182 can be referred to as "ΔRG", whereas deletions within amino acid residues corresponding to T183 and G184 can be referred to as "ΔTG".

[0074] Other mutations present in the best performing variants are as follows, using SEQ ID NO:1 for numbering: T183, E190, M202, Q172, A186 and / or I324, for example T183+E190, T183+M202, T183+E190+M202, Q172+A186+I324 and It is located at the location indicated on A186+I324.

[0075] The specific mutations present in the best performing variants are: T183D, E190P, M202L, Q172R, A186G and / or I324M, for example T183D+E190P, T183D+M202L, T183D+E190P+M202L, Q172R+A186G+I324M and Shown in A186G+I324M.

[0076] Combinations of these mutations may be used in conjunction with the above deletions at positions corresponding to R181, G182, T183 and / or G184. The substitution T183D may have a large effect on solubility but is not expected to contribute significantly to performance. The above mutations are likely to be combined with other mutations described in α-amylases, for which there is a significant amount of published patent literature.

[0077] In some embodiments, the α-amylase variants have the indicated combination of mutations and a specified degree of amino acid sequence homology / identity with SEQ ID NO:1, for example 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 homology / identity.

[0078] In some embodiments, the α-amylase variants are derived from a parent amylase having the indicated combination of mutations and a specified degree of amino acid sequence homology / identity to SEQ ID NO:1, for example 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 homology / identity.

[0079] In addition, the amylase can contain any number of conservative amino acid substitutions. Exemplary conservative amino acid substitutions are listed in Table 1. [Table 1]

[0080] The present amylases may further be derived from any of the amylase variants described above by substitution, deletion or addition of one or a few amino acids within the amino acid sequence, for example, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or even less than 2 substitutions, deletions or additions. Such variants should have the same activity as the amylase from which they are derived.

[0081] The reader will understand that some of the above conservative mutations can be generated by genetic engineering, while other conservative mutations are generated by introducing synthetic amino acids into the polypeptide by genetic or other means.

[0082] The amylase may be "precursor," "immature," or "full-length," which in turn includes a signal sequence, or "mature," which in turn lacks a signal sequence. The mature form of the polypeptide is generally the most useful. Unless otherwise specified, the numbering of amino acid residues used herein refers to the mature form of each amylase polypeptide. The amylase polypeptide may be truncated to remove the N-terminus or C-terminus, so long as the resulting polypeptide retains amylase activity.

[0083] The amylase may be a "chimeric" or "hybrid" polypeptide in that it comprises at least a portion of a first amylase polypeptide and at least a portion of a second amylase polypeptide (such chimeric amylases have been "rediscovered" in recent years as domain-swapped amylases). The amylase may further comprise a heterologous signal sequence, an epitope to allow tracking or purification, and the like. Exemplary heterologous signal sequences are from B. licheniformis amylase (LAT), B. subtilis (AmyE or AprE), and Streptomyces CelA.

[0084] 2.5. Nucleotides Encoding Variant Amylase Polypeptides In yet another aspect, nucleic acids encoding variant amylase polypeptides are provided, which may encode a particular amylase polypeptide or an amylase having a defined degree of amino acid sequence identity with a particular amylase.

[0085] In some embodiments, the nucleic acid encodes an amylase having at least 60%, at least 65%, 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% homology / identity to SEQ ID NO: 1 (excluding the portion of the nucleic acid encoding the signal sequence). It will be understood that due to the degeneracy of the genetic code, multiple nucleic acids may encode the same polypeptide.

[0086] In some embodiments, the nucleic acid hybridizes under stringent or highly stringent conditions to a nucleic acid encoding (or complementary to a nucleic acid encoding) an amylase having 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% homology / identity to SEQ ID NO:1 (excluding the portion of the nucleic acid encoding the signal sequence).

[0087] In some embodiments, the nucleic acid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or even at least 99% identity to SEQ ID NO: 2. In some embodiments, the nucleic acid hybridizes under stringent or highly stringent conditions to the nucleic acid of SEQ ID NO: 2 or to the complement of this nucleic acid. [ka]

[0088] 3. Generation of mutant amylases The variant amylases can be produced in host cells, for example, by secretion or intracellular expression, using methods well known in the art. Fermentation, separation and concentration techniques are well known in the art, and concentrated variant α-amylase polypeptide-containing solutions can be prepared using conventional methods.

[0089] To restore production scale, the variant α-amylase polypeptide can be concentrated or partially purified, generally as described above, by removing the cells via flocculation with a polymer. Alternatively, the enzyme can be concentrated or purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment. However, in some applications, the enzyme does not need to be concentrated or purified and the whole broth culture can be lysed and used without further processing. The enzyme can then be processed, for example, into granules.

[0090] 4. Compositions and Uses of Variant Amylases The mutant amylases are useful for a variety of industrial applications. For example, the mutant amylases are useful in starch conversion processes, particularly in saccharification processes of starch undergoing liquefaction. The desired end product can be any product that can be produced by enzymatic conversion of a starch substrate. For example, the desired product can be a syrup rich in glucose and maltose that can be used in other processes, such as the preparation of HFCS, or can be converted into a number of useful products, such as ascorbic acid intermediates (e.g., gluconate; 2-keto-L-gulonic acid; 5-keto-gluconic acid; and 2,5-diketogluconate); 1,3-propanediol; aromatic amino acids (e.g., tyrosine, phenylalanine, and tryptophan); organic acids (e.g., lactate, pyruvate, succinate, isocitrate, and oxaloacetate); amino acids (e.g., serine and glycine); antibiotics; antimicrobial agents; enzymes; vitamins; and hormones.

[0091] The starch conversion process may precede or be performed simultaneously with a fermentation process designed to produce alcohol for fuel or drinking alcohol (potable alcohol). Those skilled in the art will recognize the various fermentation conditions that can be used in the production of these end products. The variant amylases are also useful in food preparation compositions and methods. These various uses of the variant amylases are described in more detail below.

[0092] 4.1. Preparation of starch substrate Those skilled in the art will clearly recognize available methods that can be used to prepare starch substrates for use in the processes disclosed herein. For example, useful starch substrates can be obtained from tubers, roots, stems, legumes, cereals, or whole grains. More specifically, granular starch can be obtained from corn, corncob, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, peas, beans, bananas, or potatoes. Corn contains about 60-68% starch, barley contains about 55-65% starch, millet contains about 75-80% starch, wheat contains about 60-65% starch, and polished rice contains 70-72% starch. Particularly contemplated starch substrates are corn starch and wheat starch. Starches from cereal grains can be milled or whole, including, for example, corn solids such as kernels, bran and / or cobs. Starches can be highly refined raw starches or feedstock from starch refining processes. Various starches are also commercially available. For example, corn starch is available from Cerestar, Sigma and Katayama Chemical Industry Co. (Japan), wheat starch is available from Sigma, sweet potato starch is available from Wako Pure Chemical Industry Co. (Japan), and potato starch is available from Nakaari Chemical Pharmaceutical Co. (Japan).

[0093] The starch substrate can be crude starch from milled whole grains containing non-starch fractions such as germ residues and fiber. The milling process can include either wet milling or dry milling or grinding. In wet milling, the whole grain is soaked in water or dilute acid to separate the kernel into its components such as starch, protein, germ, oil, kernel fiber. Wet milling efficiently separates the germ and flour (i.e., starch granules and protein) and is particularly suitable for producing syrup. Approximately 90% of the corn oil is contained within the germ. In dry milling or grinding, the whole kernel is ground into a fine powder and the grain is often processed without fractionation into its components. In some cases, the oil and / or fiber from the kernel is recovered. Thus, the dry milled grain will contain a significant amount of non-starch carbohydrate compounds in addition to starch. Dry grinding of starch substrates can be used for the production of ethanol and other biochemicals. The starch to be processed may be a highly refined starch quality, for example at least 90%, at least 95%, at least 97% or at least 99.5% pure.

[0094] 4.2. Starch Gelatinization and Liquefaction The term "liquefaction" or "liquefy" as used herein refers to the process by which starch is converted to less viscous and shorter chain dextrins. Generally, the process involves gelatinization of starch, either simultaneously with or followed by the addition of α-amylase, although additional liquefaction-inducing enzymes can be optionally added. In some embodiments, the starch substrate prepared as described above is slurried with water. The starch slurry can contain starch as a weight percent dry solids of about 10-55%, about 20-45%, about 30-45%, about 30-40%, or about 30-35%. The α-amylase can be added to the slurry, for example, using a metering pump. The α-amylase typically used for this application is a thermostable bacterial α-amylase, such as Geobacillus stearothermophilus α-amylase. Alpha-amylase is typically supplied at, for example, about 1,500 units / kg (dry matter of starch). To optimize the stability and activity of the alpha-amylase, the pH of the slurry is typically adjusted to about pH 4.5-6.5, and about 1 mM calcium (about 40 ppm free calcium ion) may also be added depending on the characteristics of the amylase used. Bacterial alpha-amylase remaining in the slurry after liquefaction can be deactivated by a number of methods, including lowering the pH in a subsequent reaction step, or removing calcium from the slurry if the enzyme is calcium dependent.

[0095] The starch + α-amylase slurry may be continuously pumped through a jet cooker that is steam heated to 105°C. Gelatinization occurs rapidly under these conditions, and enzyme activity coupled with significant shear forces initiates hydrolysis of the starch substrate. The residence time in the jet cooker is short. The partially gelatinized starch is passed through a series of holding tubes maintained at 105-110°C and held for 5-8 minutes to complete the gelatinization process ("primary liquefaction"). The hydrolysis of the required DE is completed in holding tanks at elevated temperatures of 85-95°C or higher for about 1-2 hours ("secondary liquefaction"). These tanks may contain baffles to prevent backmixing. As used herein, the term "minutes of secondary liquefaction" refers to the time elapsed from the start of secondary liquefaction to the time at which the dextrose equivalent (DE) is measured. The slurry is then cooled to room temperature. The cooling step may be for a period of from 30 minutes to 180 minutes, for example from 90 minutes to 120 minutes. The liquefied starch is typically in the form of a slurry having a dry solids content (wt / wt) of about 10-50%, about 10-45%, about 15-40%, about 20-40%, about 25-40%, or about 25-35%.

[0096] Liquefaction with variant amylases can advantageously be carried out at low pH, eliminating the requirement to adjust the pH to about pH 5.5-6.5. The variant amylases can be used for liquefaction at a pH range of 2-7, e.g., pH 3.0-7.5, pH 4.0-6.0 or pH 4.5-5.8. The variant amylases can maintain liquefaction activity at a temperature range of about 85°C-95°C, e.g., 85°C, 90°C or 95°C. For example, liquefaction can be carried out with 800 μg of amylase in a 25% DS corn starch solution, e.g., at pH 5.8 and 85°C, or pH 4.5 and 95°C for 10 minutes. Liquefaction activity can be assayed using any of a number of viscosity assays known in the art.

[0097] In certain embodiments using the present amylase variants, starch liquefaction is carried out at temperatures ranging from 90-115° C. to produce, for example, high purity glucose syrup, HFCS, maltodextrins, and the like.

[0098] 4.3. Saccharification The liquefied starch can be saccharified using the variant amylase, optionally in the presence of another enzyme, to a syrup rich in low DP (e.g. DP1+DP2) sugars. The exact composition of the product of saccharification depends on the enzyme combination used and the type of granular starch processed. Advantageously, the syrup obtainable using the provided variant amylases may contain a weight % of DP2 of more than 30%, e.g. 45%-65% or 55%-65% of the total oligosaccharides in the saccharified starch. The weight % of (DP1+DP2) in the saccharified starch may be more than about 70%, e.g. 75%-85% or 80%-85%. The amylases also result in a fairly high yield of DP1 of glucose in the syrup product, e.g. more than 20%.

[0099] Liquefaction is generally carried out as a continuous process, whereas saccharification is often carried out as a batch process. Saccharification is typically most effective at a temperature of about 60-65°C and a pH of about 4.0-4.5, e.g. pH 4.3, making it necessary to cool the liquefied starch and adjust the pH. The temperature and pH ranges may vary depending on the properties of the enzyme. Saccharification can be carried out at temperatures of, e.g., about 40°C, about 50°C or about 55°C to about 60°C or about 65°C. Saccharification is usually carried out in stirred tanks, which can take several hours to fill or empty. The enzyme is typically added either at a fixed ratio to the dry solids when the tank is filled, or as a one-time addition at the start of the filling stage. Saccharification reactions to produce syrups are typically carried out for about 24-72 hours, e.g., 24-48 hours. Once the maximum or desired DE is reached, the reaction is stopped, e.g., by heating to 85°C for 5 minutes. Subsequent incubation will result in a lower DE, eventually up to about 90 DE, as the accumulated glucose repolymerizes into isomaltose and / or other reversion products using enzymatic reversion reactions and / or thermodynamic equilibrium approaches. When using amylase, saccharification is optimally carried out at a temperature range of about 30° C. to about 75° C., e.g., 45° C. to 75° C. or 47° C. to 74° C. The saccharification step may be carried out over a pH range of about pH 3 to about pH 7, e.g., pH 3.0 to pH 7.5, pH 3.5 to pH 5.5, pH 3.5, pH 3.8 or pH 4.5.

[0100] The α-amylase may be added to the slurry in the form of a composition. The α-amylase may be added to the slurry of granular starch substrate in an amount of about 0.6-10 ppm (ds), e.g., 2 ppm (ds). The α-amylase may be added as whole broth, clarified, concentrated, partially purified, or purified enzyme. The specific activity of the amylase may be about 300 U / mg enzyme, e.g., as measured using the PAHBAH assay. The α-amylase may also be added as a whole broth product.

[0101] The α-amylase may be added to the slurry as an isolated enzyme solution. For example, the α-amylase may be added in the form of cultured cell material produced by a host cell expressing the amylase. The α-amylase may be secreted by the host cell into the reaction medium during the fermentation or SSF process so that the enzyme is continuously provided within the reaction. The host cell producing and secreting the amylase may also express additional enzymes, such as glucoamylase. For example, U.S. Pat. No. 5,422,267 discloses the use of glucoamylase in yeast in the production of alcoholic beverages. For example, a host cell, such as Trichoderma reesei or Aspergillus niger, may be modified to co-express α-amylase and glucoamylase, such as HgGA, TrGA or TrGA variants, during saccharification. The host cell may be genetically engineered not to express its endogenous glucoamylase and / or other enzymes, proteins or other substances. Host cells can be engineered to express a wide variety of glycolytic enzymes. For example, recombinant yeast host cells can include nucleic acids encoding glucoamylase, α-glucosidase, enzymes that utilize pentose sugars, α-amylase, pullulanase, isoamylase, and / or isopullulanase. See, e.g., WO 2011 / 153516 A2.

[0102] 4.4. Isomerization The soluble starch hydrolysate produced by treatment with amylase can be converted to high fructose starch-based syrup (HFSS), e.g., high fructose corn syrup (HFCS). This can be accomplished using glucose isomerase, particularly glucose isomerase immobilized on a solid support. The pH is raised to about 6.0 to about 8.0 (depending on the isomerase), e.g., pH 7.5, and Ca is added. 2+is removed by ion exchange. Suitable isomerases include SWEETZYME®, IT (Novozymes A / S); G-ZYME® IMGI, as well as G-ZYME® G993, KETOMAX®, G-ZYME® G993, G-ZYME® G993 Liquid, and GENSWEET® IGI. After isomerization, the mixture typically contains about 40-45% fructose, for example 42% fructose.

[0103] Fermentation Soluble starch hydrolysates, especially high glucose syrups, can be fermented by contacting the starch hydrolysates with a fermenting organism, typically at a temperature of about 32° C., e.g., 30° C. to 35° C. for alcohol-producing yeasts. The temperature and pH of the fermentation will depend on the fermenting organism. EOF products include metabolic products such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono-Δ-lactone, sodium erythorbate, lysine and other amino acids, Ω3 fatty acids, butanol, isoprene, 1,3-propanediol, and other biomaterials.

[0104] Ethanol-producing microorganisms include yeasts, such as Saccharomyces cerevisiae, and bacteria, such as Zymomonas moblis, which express alcohol dehydrogenase and pyruvate decarboxylase. Ethanol-producing microorganisms can express xylose reductase and xylitol dehydrogenase, which convert xylose to xylulose. For example, improved strains of ethanol-producing microorganisms that can withstand high temperatures are known in the art and can be used. See Liu et al. (2011) Sheng Wu Gong Cheng Xue Bao 27:1049-56. Commercial sources of yeast include ETHANOL RED® (LeSaffre); FERMAX™ (Martrex), THERMOSACC® (Lallemand); RED STAR® (Red Star); FERMIOL® (DSM Specialties); and SUPERSTART® (Alltech). Microorganisms that produce other metabolic products by fermentation, such as citric acid and lactic acid, are also known in the art. See, e.g., Papagianni (2007) Biotechnol. Adv. 25:244-63; John et al. (2009) Biotechnol. Adv. 27:145-52.

[0105] The saccharification and fermentation process may be carried out as an SSF process. Fermentation may include, for example, subsequent concentration, purification and recovery of ethanol. During fermentation, the ethanol content of the broth or "beer" may reach about 8-18% v / v, e.g., 14-15% v / v. The broth may be distilled to produce a concentrated, e.g., 96% pure, solution of ethanol. Additionally, the CO2 produced by fermentation may be collected using a CO2 scrubber, compressed, and marketed for other uses, e.g., for the production of carbonated beverages or dry ice. Solid waste from the fermentation process may be used as a high protein product, e.g., livestock feed.

[0106] As mentioned above, the SSF process can be carried out using fungal cells that continuously express and secrete amylase throughout the SSF. The fungal cells expressing amylase can also be fermenting microorganisms, e.g., ethanol-producing microorganisms. Thus, ethanol production can be carried out using fungal cells that express sufficient amylase so that there is little need to add exogenous enzymes. The fungal host cells can be from appropriately modified fungal strains. In addition to amylase, fungal host cells that express and secrete other enzymes can also be used. Such cells may express glucoamylase and / or pullulanase, phytase, α-glucosidase, isoamylase, β-amylase cellulase, xylanase, other hemicellulases, proteases, β-glucosidase, pectinases, esterases, oxidoreductases, transferases, or other enzymes.

[0107] A variation of this process is the "fed-batch fermentation" system, where substrate is added in small increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression can inhibit the metabolism of cells and when it is desirable to have limited amounts of substrate in the medium. The actual substrate concentration in a fed-batch system is estimated by changes in measurable factors such as pH, dissolved oxygen, and partial pressure of exhaust gases such as CO2. Batch and fed-batch fermentation are common and well known in the art.

[0108] Continuous fermentation is an open system in which a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant high density where the cells are primarily in log phase growth. Continuous fermentation allows for the regulation of cell growth and / or product concentration. For example, the limiting nutrient factor, such as the carbon or nitrogen source, is maintained at a constant ratio and all other parameters are allowed to adjust. Since growth is maintained at steady state, cell loss due to medium being removed must be balanced against the cell growth rate during fermentation. Methods for optimizing continuous fermentation processes to maximize the rate of product formation are well known in the field of industrial microbiology.

[0109] 4.6. Compositions Comprising Variant Amylases The variant amylase can be combined with a glucoamylase (EC 3.2.1.3), such as a Trichoderma glucoamylase or variant thereof. An exemplary glucoamylase is Trichoderma reesei glucoamylase (TrGA) and variants thereof with superior specific activity and thermostability. See US Patent Publication Nos. 2006 / 0094080, 2007 / 0004018 and 2007 / 0015266 (Danisco US Inc.). Suitable variants of TrGA include those with glucoamylase activity and at least 80%, at least 90% or at least 95% sequence identity with wild-type TrGA. The variant amylase advantageously increases the yield of glucose produced in the saccharification process catalyzed by TrGA.

[0110] Alternatively, the glucoamylase can be another glucoamylase derived from a plant (including algae), a fungus, or a bacteria. For example, the glucoamylase can be an Aspergillus niger G1 or G2 glucoamylase or a variant thereof (see, e.g., Boel et al. (1984) EMBO J. 3:1097-1102; WO 92 / 00381; WO 00 / 04136 (Novo Nordisk A / S)); and an A. awamori glucoamylase (see, e.g., WO 84 / 02921 (Cetus Corp.)). Other contemplated Aspergillus glucoamylases include mutants with enhanced thermostability, such as G137A and G139A (Chen et al. (1996) Prot. Eng. 9:499-505); D257E and D293E / Q (Chen et al. (1995) Prot. Eng. 8:575-582); N182 (Chen et al. (1994) Biochem. J. 301:275-281); A246C (Fierobe et al. (1996) Biochemistry 35:8698-8704); and mutants containing Pro residues at positions A435 and S436 (Li et al. (1997) Protein Eng. 10:1199-1204). Other contemplated glucoamylases include Talaromyces glucoamylases derived from T. emersonii (e.g., WO 99 / 28448, Novo Nordisk A / S), T. leycettanus (e.g., U.S. Re. No. 32,153, CPC International, Inc.), T. duponti, or T. thermophilus (e.g., U.S. Pat. No. 4,587,215), among others.Contemplated bacterial glucoamylases include those from the genus Clostridium, particularly C. thermoamylolyticum (e.g., EP 135,138 (CPC International, Inc.) and C. thermohydrosulfuricum (e.g., WO 86 / 01831 (Michigan Biotechnology Institute)). Suitable glucoamylases include those from Aspergillus oryzae, such as those described in WO 00 / 04136 (Novo Nordisk A / S). Also suitable are commercially available glucoamylases, such as AMG 200L; AMG 300L; SAN™ SUPER and AMG™ E (Novozymes); OPTIDEX™ 300 and OPTIDEX L-400 (Danisco US Inc.); AMIGASE™ and AMIGASE™ PLUS (DSM); G-ZYME™ G900 (Enzyme Bio-Systems); and G-ZYME™ G990 ZR (A. niger glucoamylase with low protease content).Still other suitable glucoamylases include Aspergillus fumigatus glucoamylase, Talaromyces sp. glucoamylase, Thielavia sp. glucoamylase, Trametes sp. glucoamylase, Thermomyces sp. glucoamylase, Athelia sp. glucoamylase, Humicola sp. glucoamylase (e.g., HgGA), Penicillium sp. glucoamylase, Artomyces sp. glucoamylase, Gloeophyllum sp. glucoamylase, Pycnoporus sp. glucoamylase, or Stecherinum sp. glucoamylase. Glucoamylase is typically added in an amount of about 0.1 to 2 glucoamylase units (GAU) / g(ds), for example about 0.16 GAU / g(ds), 0.23 GAU / g(ds) or 0.33 GAU / g(ds).

[0111] Other suitable enzymes that can be used together with amylase include phytases, proteases, pullulanases, β-amylases, isoamylases, different α-amylases, α-glucosidases, cellulases, xylanases, other hemicellulases, β-glucosidases, transferases, pectinases, lipases, cutinases, esterases, oxidoreductases or combinations thereof. For example, debranching enzymes such as isoamylase (EC 3.2.1.68) can be added in effective amounts well known to those skilled in the art. Pullulanases (EC 3.2.1.41), such as PROMOZYME®, are also suitable. Pullulanases are typically added at 100 U / kg (ds). Other suitable enzymes include proteases, such as fungal proteases and bacterial proteases. Fungal proteases include those obtained from the genus Aspergillus, e.g., A. niger, A. awamori, A. oryzae; Mucor (e.g., M. miehei); Rhizopus; and Trichoderma.

[0112] β-Amylases (EC 3.2.1.2) are exo-acting malt-producing amylases that catalyze the hydrolysis of 1,4-α-glucosidic bonds in amylopectin and related glucose polymers, thereby releasing maltose. β-Amylases have been isolated from a variety of plants and microorganisms. See Fogarty et al. (1979) in Progress in Industrial Microbiology, Vol. 15, pp. 112-115. The optimum temperature for these β-amylases is in the range of 40° C. to 65° C., and the optimum pH is in the range of about 4.5 to about 7.0. Contemplated β-amylases include, but are not limited to, β-amylases derived from barley SPEZYME® BBA 1500, SPEZYME® DBA, OPTIMALT™ ME, OPTIMALT™ BBA (Danisco US Inc.); and NOVOZYM™ WBA (Novozymes A / S).

[0113] Compositions comprising the present amylases may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., which may further comprise any one or more of the additional enzymes listed herein, along with buffers, salts, preservatives, water, co-solvents, surfactants, etc. Such compositions may work in combination with endogenous enzymes or other components already present in the slurry, water bath, washing machine, food or beverage product, etc., such as endogenous plant (including algae) enzymes, residual enzymes from prior processing steps, etc.

[0114] 5. Compositions and methods for baking and food preparation The present invention also relates to "food compositions" including, but not limited to, foods, animal feeds and / or food / feed additives comprising the amylase and methods for preparing such food compositions or uses thereof comprising combining the variant amylase with one or more food ingredients.

[0115] Furthermore, the present invention relates to the use of an amylase in the preparation of a food composition, which is baked following the addition of a polypeptide of the present invention. As used herein, the term "baking composition" refers to a composition and / or additive prepared in the process of providing a baked food product, including but not limited to bread flour, dough, baking additives and / or baked products. The food composition or additive may be liquid or solid.

[0116] 6. Fabric desizing compositions and uses Also contemplated are compositions and methods for treating textiles (e.g., desizing fabrics) using amylase. Methods for treating textiles are well known in the art (see, e.g., U.S. Pat. No. 6,077,316). For example, the feel and appearance of textiles can be improved by a method that includes contacting the textile with an amylase in a solution. The textile can be treated with the solution under pressure.

[0117] Amylases can be applied during or after weaving of the textile, during the desizing stage or one or more additional textile processing steps. During weaving of textiles, the yarns are subjected to fairly strong mechanical strain. Before weaving on the loom, the warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. Amylases can be applied during or after weaving to remove these sizing starches or starch derivatives. After weaving, amylases can be used to remove the sizing coating before further processing of the textile to ensure a homogeneous and wash-resistant result.

[0118] Amylases can be used alone or with other desizing chemicals and / or desizing enzymes to desize textiles, including cotton-containing textiles, as detergent additives, for example in aqueous compositions. Amylases can also be used in compositions and methods for creating a stonewashed look on indigo dyed denim textiles and garments. To produce garments, textiles can be cut and sewn into garments or garments that are subsequently finished. Various enzymatic finishing methods have been developed, particularly for producing denim jeans. Finishing denim garments usually begins with enzymatic desizing, during which the garment is subjected to the action of proteolytic enzymes to provide softness to the textile and to make the cotton more amenable to subsequent enzymatic finishing steps. Amylases can be used in denim garment finishing (e.g., "bio-stone processing"), enzymatic desizing and softening to the textile and / or finishing processes.

[0119] 7. Cleaning Compositions One embodiment of the compositions and methods is a cleaning composition comprising an amylase as a component. The amylase polypeptide can be used as a component in detergent compositions for, for example, hand washing, washing machine washing, dish washing, and cleaning other hard surfaces. Such compositions include heavy duty liquid (HDL), heavy duty dry (HDD) and hand (manual) laundry detergent compositions, including laundry detergent compositions in unit dose format, and automatic dishwashing (ADW) and hand (manual) dishwashing compositions, including dishwashing compositions in unit dose format.

[0120] Overview Preferably, the amylase is incorporated into the detergent at or near the concentration conventionally used for amylase in detergents. For example, the amylase polypeptide may be added in an amount equivalent to 0.00001-1 mg of amylase (calculated as pure enzyme protein) per liter of wash / dishwashing liquor. Exemplary preparations are provided herein, as exemplified below.

[0121] The amylase polypeptide may be a component of the detergent composition as the sole enzyme or together with other enzymes, including other amylolytically active enzymes. Thus, the amylase polypeptide may be included in the detergent composition in the form of non-dusted granules, stabilized liquids or protected enzymes. Non-dusted granules may be prepared, for example, as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452, and may be optionally coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycols, PEGs) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and further 15 to 80 ethylene oxide units are present; fatty alcohols; fatty acids; mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are described, for example, in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid according to established methods. Other enzyme stabilizers are known in the art. Protected enzymes can be prepared, for example, according to the methods disclosed in EP 238216. Polyols have been recognized for many years as protein stabilizers, as well as improving protein solubility.

[0122] The detergent composition may be in any useful form, such as, for example, powder, granule, paste, bar, or liquid. Liquid detergents may typically be aqueous, containing up to about 70% water and 0% to 30% organic solvent. Liquid detergents may also be in the form of compact gels, containing only about 30% water.

[0123] The detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic or zwitterionic. The detergent will typically contain 0% to about 50% of anionic surfactants, such as linear alkylbenzene sulfonates (LAS); alpha-olefin sulfonates (AOS); alkyl sulfates (fatty alcohol sulfates) (AS); alcohol ethoxy sulfates (AEOS or AES); secondary alkane sulfonates (SAS); alpha-sulfo fatty acid methyl esters; alkyl or alkenyl succinic acids; or soaps. The composition may also contain 0% to about 40% of nonionic surfactants, such as alcohol ethoxylates (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethylamine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides or polyhydroxyalkyl fatty acid amides (described, for example, in WO 92 / 06154).

[0124] The detergent composition may additionally contain one or more other enzymes, such as a protease, another starch degrading enzyme, cutinase, lipase, cellulase, pectate lyase, perhydrolase, xylanase, peroxidase and / or laccase, in any combination.

[0125] The detergent may contain from about 1% to about 65% of detergent builders or complexing agents, such as, for example, zeolites, diphosphates, triphosphates, phosphonates, citrates, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl and alkenyl succinic acids, soluble or layered silicates (e.g., SKS-6 from Hoechst). The detergent may be unbuilt, i.e., substantially free of detergent builders. The enzymes may be used in any composition compatible with the stability of the enzyme. Enzymes may generally be protected from harmful components by known forms of encapsulation, such as by granulation or sequestration in hydrogels. Enzymes, and particularly amylases, with or without starch binding domains, may be used in a variety of compositions, including laundry and dishwashing applications, surface cleaners, and compositions for ethanol production from starch or biomass.

[0126] The detergent may include one or more polymers, examples of which include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers, and methacrylic acid / lauryl acrylate copolymers.

[0127] The detergent may contain a bleaching system which may be combined with a peracid forming bleach activator such as, for example, tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS) and may include a H2O2 source such as, for example, a perborate or percarbonate. Alternatively, the bleaching system may include a peroxyacid (e.g., an amide-, imide- or sulfone-type peroxyacid). The bleaching system may be an enzymatic bleaching system, for example, a perhydrolase as described in WO 2005 / 056783.

[0128] The enzymes of the detergent compositions may be stabilized using conventional stabilizers, for example polyols such as propylene glycol or glycerol; sugars or sugar alcohols; lactic acid; boric acid or boric acid derivatives such as aromatic boric acid esters, and the compositions may be prepared, for example, as described in WO 92 / 19709 and WO 92 / 19708.

[0129] The detergent may also contain other conventional detergent ingredients such as clays, foam boosters, suds suppressors, anti-corrosive agents, soil suspending agents, soil anti-redeposition agents, dyes, disinfectants, anti-tarnish agents, optical brighteners or fabric softeners including fragrances.

[0130] The pH (measured in an aqueous solution at the concentration used) is usually neutral or alkaline, for example, a pH of about 7.0 to about 11.0.

[0131] The following describes specific forms of detergent compositions for incorporating the α-amylase. Many of these compositions can be provided in unit dose format for ease of use. Unit dose preparations and packaging are described, for example, in U.S. Patent Publication Nos. 20090209445A1, 20100081598A1, U.S. Patent No. 7001878B2, EP1504994B1, WO2001085888A2, WO2003089562A1, WO2009098659A1, and the like. Brochure No. 2009098660A1, Brochure No. 2009112992A1, Brochure No. 2009124160A1, Brochure No. 2009152031A1, Brochure No. 2010059483A1, Brochure No. 2010088112A1, Brochure No. 2010090915A1, Brochure No. 201 Brochure No. 0135238A1, Brochure No. 2011094687A1, Brochure No. 2011094690A1, Brochure No. 2011127102A1, Brochure No. 2011163428A1, Brochure No. 2008000567A1, Brochure No. 2006045391A1, Brochure No. 2006007911A1 No. 2012027404A1, EP 1740690B1, WO 2012059336A1, U.S. Pat. No. 6,730646B1, WO 2008087426A1, WO 2010116139A1, and WO 2012104613A1.

[0132] 7.2. Heavy Duty Liquid (HDL) Laundry Detergent Compositions A typical HDL laundry detergent composition comprises a detersive surfactant (10% to 40% w / w) including an anionic detersive surfactant (selected from the group of linear, branched or random chain substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkoxylated alkyl sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and / or mixtures thereof) and, optionally, a nonionic surfactant (selected from the group of linear, branched or random chain substituted or unsubstituted alkoxylated alkyl alcohols, such as C8-C18 ethoxylated alkyl alcohols and / or C6-C12 alkylphenol alkoxylates), wherein the weight ratio of the detersive anionic surfactant (having a hydrophilicity index (HIc) of 6.0 to 9) to the detersive nonionic surfactant is greater than 1:1. Suitable detersive surfactants also include cationic detersive surfactants (selected from the group of alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary sulfonium compounds and / or mixtures thereof); zwitterionic and / or amphoteric detersive surfactants (selected from the group of alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants, and mixtures thereof.

[0133] The composition may optionally comprise a surfactant enhancing polymer consisting of an amphiphilic alkoxylated grease cleaning polymer (selected from the group of branched hydrophilic and hydrophobic alkoxylated polymers, e.g. alkoxylated polyalkyleneimines in the range of 0.05% to 10% by weight) and / or a random graft polymer (typically comprising a hydrophilic backbone comprising monomers selected from the group consisting of unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyalcohols, e.g. glycerol and mixtures thereof; and a hydrophobic side chain selected from the group consisting of C4-C25 alkyl groups, polypropylene, polybutylene, vinyl esters of saturated C1-C6 mono-carboxylic acids, C1-C6 alkyl esters of acrylic or methacrylic acid and mixtures thereof).

[0134] The composition may, for example, be a soil repellent polymer (non-ionic end-capped polyesters, such as SRP1, polymers comprising at least one monomer unit selected from sugars, dicarboxylic acids, polyols and combinations thereof in a random or block structure, ethylene terephthalate based polymers and copolymers thereof in a random or block structure, such as Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, Marloquest SL), anti-redeposition polymers (0.1% to 10% by weight, in the molecular weight range of 500 to 100,000 Da, including carboxylic acid polymers such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixtures thereof, vinylpyrrolidone homopolymer, and / or polyethylene glycol); cellulose polymers (including polymers selected from alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, examples of which include alkyl carboxyalkyl celluloses, including carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixtures thereof), and polymeric carboxylates (e.g., maleic acid / acrylic acid random copolymers or polyacrylate homopolymers).

[0135] The composition may further comprise saturated or unsaturated fatty acids, preferably saturated or unsaturated C12-C24 fatty acids (0% to 10% by weight); deposition aids (examples of which include polysaccharides, preferably cellulose polymers, polydiallyldimethylammonium halides (DADMAC) and copolymers of DAD MAC in random or block configuration with vinylpyrrolidone, acrylamide, imidazole, imidazolinium halides and mixtures thereof, cationic guar gum, cationic celluloses such as cationic hydroxyethylcellulose, cationic starch, cationic polyacrylamide and mixtures thereof).

[0136] The compositions may also include dye transfer inhibitors, examples of which include manganese phthalocyanine, peroxidase, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone and polyvinylimidazole and / or mixtures thereof; examples of which include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentamethylenephosphonic acid (DTPMP), hydroxyethanediphosphonic acid (HEDP), ethylenediamine N,N'-disuccinic acid (EDDS), methylglycine diacetate (MGDA), diethylenetriaminepentaacetic acid (DTPA), propylenediaminetetraacetic acid (PDTA ), 2-hydroxypyridine-N-oxide (HPNO) or methylglycine diacetate (MGDA), glutamic acid N,N-diacetate (N,N-dicarboxymethylglutamic acid tetrasodium salt (GLDA), nitrilotriacetic acid (NTA), 4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any salts thereof, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and derivatives thereof.

[0137] The composition preferably comprises an enzyme selected from protease, amylase, lipase, cellulase, choline oxidase, peroxidase / oxidase, pectate lyase, mannanase, cutinase, laccase, phospholipase, lysophospholipase, acyltransferase, perhydrolase, arylesterase, and any mixture thereof (generally about 0.01% to 0.03% by weight of active enzyme). The composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or a boric acid derivative, such as an aromatic borate ester or a phenylboronic acid derivative, such as 4-formylphenylboronic acid).

[0138] The composition optionally includes silicone-based or fatty acid-based suds suppressors; hueing dyes, calcium and magnesium cations, visual signal components, antifoam agents (0.001% to about 4.0% by weight), and / or structurants / thickeners (0.01% to about 5% by weight selected from the group consisting of di- and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulosic materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof).

[0139] The composition can be in any liquid form, such as a liquid or gel form or any combination thereof. The composition can be in any unit dose form, such as a pouch.

[0140] 7.3. Heavy Duty Dry / Solid (HDD) Laundry Detergent Compositions Exemplary HDD laundry detergent compositions include detersive surfactants including anionic detersive surfactants (e.g., linear or branched or random chain substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof), nonionic detersive surfactants (e.g., linear or branched or random chain substituted or unsubstituted C8-C18 alkyl ethoxylates and / or C6-C12 alkyl phenol alkoxylates), cationic detersive surfactants (e.g., alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary sulfonium compounds, and mixtures thereof), zwitterionic and / or amphoteric detersive surfactants (e.g., alkanolamine sulfobetaines), amphoteric surfactants, semi-polar nonionic surfactants, and mixtures thereof); non-phosphate containing builders (e.g., examples of which include zeolite A, zeolite X in the range of 0% to less than 10% by weight). , zeolite builders including zeolite P and zeolite MAP), phosphate builders (e.g., sodium tripolyphosphate in the range of 0% to less than 10% by weight), citric acid, citrates and nitrilotriacetic acid, silicates (e.g., sodium or potassium silicate or sodium metasilicate in the range of 0% to less than 10% by weight, or layered silicate (SKS-6)); carbonates (e.g., sodium carbonate and / or sodium bicarbonate in the range of 0% to less than 80% by weight); and photobleaching. Colorants (e.g., sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes and mixtures thereof), hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED, nonanoyloxybenzenesulfonate-NOBS, nitrile quats and mixtures thereof);A source of hydrogen peroxide (e.g., inorganic perhydrate salts, examples of which include sodium salts of perborate, percarbonate, persulfate, perphosphate, or persilicate, monohydrate or tetrahydrate), preformed hydrophilic and / or hydrophobic peracids (e.g., percarboxylic acids and salts, percarbonates and salts, perimidic acids and salts, peroxymonosulfates and salts, and mixtures thereof), and / or bleach catalysts (e.g., imine bleach boosters, examples of which include iminium cations and polyions, iminium zwitterions, modified amines, modified amine ions ... oxides, N-sulfonylimines, N-phosphonylimines, N-acylimines, thiadiazole dioxides, perfluoroimines, cyclic sugar ketones and mixtures thereof, and bleaching agents containing metal-containing bleach catalysts (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations, together with auxiliary metal cations, such as zinc or aluminum, and sequestering agents, such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and the water-soluble salts thereof);

[0141] The composition preferably comprises an enzyme such as a protease, amylase, lipase, cellulase, choline oxidase, peroxidase / oxidase, pectate lyase, mannanase, cutinase, laccase, phospholipase, lysophospholipase, acyltransferase, perhydrolase, arylesterase and mixtures thereof.

[0142] The compositions may optionally include additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accords, hueing agents, additional polymers including fabric integrity and cationic polymers, dielock components, fabric softeners, brighteners (e.g., CI optical brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids, and / or cyclodextrins.

[0143] 7.4. Automatic Dishwashing (ADW) Detergent Compositions As discussed above, the present α-amylase variants are particularly effective in ADW applications. A typical ADW detergent composition may contain nonionic surfactants, including ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohol or amine oxide surfactants, present in amounts of 0-10% by weight; phosphate builders (e.g., monophosphates, diphosphates, tripolyphosphates, other oligomeric polyphosphates, sodium tripolyphosphate - STPP) and non-phosphate containing builders (e.g., methyl-glycine-diacetate (MGDA) and its salts and derivatives, glutamic acid-N,N-diacetate (GLDA) and its salts in the range of 0.5% to 50% by weight). and derivatives, iminodisuccinic acid (IDS) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), B-alanine diacetic acid (B-ADA) and their salts, homopolymers and copolymers of poly-carboxylic acids and their partially or fully neutralized salts, amino acid based compounds including monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts) in the range of 5-60%; sulfonated / carboxylated polymers in the range of about 0.1% to about 50% by weight to provide dimensional stability; and about 0.drying aids in the range of 1% to about 10% by weight (e.g. polyesters, especially anionic polyesters, optionally together with further monomers having another 3-6 functionality - typically acid, alcohol or ester functional groups promoting polycondensation, polycarbonate, polyurethane and / or polyurea polyorganosiloxane compounds or their precursor compounds, especially reactive cyclic carbonate and urea type precursor compounds); silicates (including sodium or potassium silicates, e.g. sodium disilicate, sodium metasilicate and crystalline phyllosilicates) in the range of about 1% to about 20% by weight; inorganic bleaches (e.g. perhydrate salts, e.g. perborates, percarbonates, perphosphates, persulfates and persilicates) and organic bleaches (e.g. organic peroxyacids including diacyl and tetraacyl peroxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid and diperoxyhexadecanedioic acid); bleach activators (i.e. organic peracid precursors in the range of about 0.1% to about 10% by weight); bleach catalysts (e.g., triazacyclononane manganese and related complexes, Co, Cu, Mn and Fe bispyridylamine and related complexes, and pentamine cobalt(III) acetate and related complexes); metal care agents (e.g., benzatriazoles, metal salts and complexes and / or silicates) in the range of about 0.1% to 5% by weight; enzymes (e.g., proteases, amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases and mixtures thereof) in the range of about 0.01 to 5.0 mg of active enzyme per gram of automatic dishwasher detergent composition; and enzyme stabilizer ingredients (e.g., oligosaccharides, polysaccharides and inorganic divalent metal salts).

[0144] 7.5. Additional Detergent Compositions Below, additional exemplary detergent formulations to which the present amylases can be added are described in numbered paragraphs.

[0145] 1) (calculated as acid) from about 7% to about 12% linear alkylbenzene sulfonate; from about 1% to about 4% alcohol ethoxy sulfate (e.g., C12-18 alcohol, 1-2 ethylene oxide (EO)) or alkyl sulfate (e.g., C16-18); from about 5% to about 9% alcohol ethoxylate (e.g., C14-15 alcohol, 7EO); from about 14% to about 20% sodium carbonate; from about 2 to about 6% soluble silicate; from about 15% to about 22% zeolite; from 0% to about 6% sodium sulfate; from about 0% to about A detergent composition formulated as granules having a bulk density of at least 600 g / L comprising: 15% sodium citrate / citric acid; about 11% to about 18% sodium perborate; about 2% to about 6% TAED; 0% to about 2% carboxymethylcellulose (CMC); 0-3% polymer (e.g., maleic acid / acrylic acid, copolymer, PVP, PEG); 0.0001-0.1% protein enzyme (calculated as pure enzyme); and 0-5% minor ingredients (e.g., foam inhibitors, fragrances, optical brighteners, photobleachers).

[0146] 2) (calculated as acid) from about 6% to about 11% linear alkylbenzene sulfonate; from about 1% to about 3% alcohol ethoxy sulfate (e.g., C12-18 alcohol, 1-2EO)) or alkyl sulfate (e.g., C16-18); from about 5% to about 9% alcohol ethoxylate (e.g., C14-15 alcohol, 7EO); from about 15% to about 21% sodium carbonate; from about 1% to about 4% soluble silicate; from about 24% to about 34% zeolite; from about 4% to about 10% of sodium sulfate (e.g., Na2SO4); 0% to about 15% sodium citrate / citric acid; 0% to about 2% carboxymethylcellulose (CMC); 1 to 6% polymer (e.g., maleic acid / acrylic acid copolymer, PVP, PEG); 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g., foam suppressors, fragrances), prepared as granules having a bulk density of at least 600 g / L.

[0147] 3) (calculated as acid) from about 5% to about 9% linear alkylbenzene sulfonates; from about 7% to about 14% alcohol ethoxylates (e.g., C12-15 alcohols, 7EO); from about 1 to about 3% fatty acids (e.g., C16-22 fatty acids) as soaps; from about 10% to about 17% sodium carbonate; from about 3 to about 9% soluble silicates; from about 23% to about 33% zeolites; from 0% to about 4% sodium sulfate; from about 8% to about 16% sodium perborate; from about 2% to about 8% of TAED; 0% to about 1% of a phosphonate (e.g., EDTMPA); 0% to about 2% of carboxymethylcellulose (CMC); 0-3% of a polymer (e.g., maleic acid / acrylic acid copolymer, PVP, PEG); 0.0001-0.1% of an enzyme (calculated as pure enzyme protein); and 0-5% of minor ingredients (e.g., foam inhibitors, fragrances, optical brighteners), the detergent composition being formulated as a granule having a bulk density of at least 600 g / L.

[0148] 4) A detergent composition prepared as a granule having a bulk density of at least 600 g / L, comprising (calculated as acid) about 8% to about 12% linear alkylbenzene sulfonate; about 10% to about 25% alcohol ethoxylate (e.g., C12-15 alcohol, 7EO); about 14% to about 22% sodium carbonate; about 1% to about 5% soluble silicate; about 25% to about 35% zeolite; 0% to about 10% sodium sulfate; 0% to about 2% carboxymethylcellulose (CMC); 1 to 3% polymer (e.g., maleic acid / acrylic acid copolymer, PVP, PEG); 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g., foam suppressors, fragrances).

[0149] 5) (calculated as acid) from about 15% to about 21% linear alkylbenzene sulfonate; from about 12% to about 18% alcohol ethoxylate (e.g., C12-15 alcohol, 7EO or C12-15 alcohol, 5EO); from about 3% to about 13% fatty acid (e.g., oleic acid) as soap; from 0% to about 13% alkenyl succinic acid (C12-14); from about 8% to about 18% aminoethanol; from about 2 0% to about 3% citric acid; 0% to about 3% phosphonate; 0% to about 3% polymer (e.g., PVP, PEG); 0% to about 2% borate; 0% to about 3% ethanol; about 8% to about 14% propylene glycol; 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g., dispersants, foam suppressors, fragrances, optical brighteners).

[0150] 6) (calculated as acid) about 15% to about 21% linear alkylbenzene sulfonate; 3 to 9% alcohol ethoxylate (e.g., C12-15 alcohol, 7EO, or C12-15 alcohol, 5EO); about 3% to about 10% fatty acid (e.g., oleic acid) as soap; about 14% to about 22% zeolite; about 9% to about 18% potassium citrate; 0% to about 2% borate; 0% to about 2% carboxymethylcellulose. an aqueous structured liquid detergent composition comprising: carbon monoxide (CMC); 0% to about 3% polymer (e.g., PEG, PVP); 0% to about 3% anchoring polymer, e.g., lauryl methacrylate / acrylic acid copolymer; molar ratio 25:1, MW 3800; 0% to about 5% glycerol; 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g., dispersants, foam suppressors, fragrances, optical brighteners).

[0151] 7) A detergent composition prepared as a granule having a bulk density of at least 600 g / L, comprising about 5% to about 10% fatty alcohol sulfate; about 3% to about 9% ethoxylated fatty acid monoethanolamide; 0 to 3% soap as fatty acid; about 5% to about 10% sodium carbonate; about 1% to about 4% soluble silicate; about 20% to about 40% zeolite; about 2% to about 8% sodium sulfate; about 12% to about 18% sodium perborate; about 2% to about 7% TAED; about 1% to about 5% polymer (e.g., maleic acid / acrylic acid copolymer, PEG); 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor components (e.g., optical brightener, foam suppressor, fragrance).

[0152] 8) A detergent composition prepared as a granule containing about 8% to about 14% linear alkylbenzene sulfonate (calculated as acid); about 5% to about 11% ethoxylated fatty acid monoethanolamide; 0% to about 3% soap as fatty acid; about 4% to about 10% sodium carbonate; about 1% to about 4% soluble silicate; about 30% to about 50% zeolite; about 3% to about 11% sodium sulfate; about 5% to about 12% sodium citrate; about 1% to about 5% polymer (e.g., PVP, maleic acid / acrylic acid copolymer, PEG); 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g., foam suppressor, fragrance).

[0153] 9) A detergent composition prepared as a granule containing about 6% to about 12% linear alkylbenzene sulfonate (calculated as acid); about 1% to about 4% nonionic surfactant; about 2% to about 6% soap as fatty acid; about 14% to about 22% sodium carbonate; about 18% to about 32% zeolite; about 5% to about 20% sodium sulfate; about 3% to about 8% sodium citrate; about 4% to about 9% sodium perborate; about 1% to about 5% bleach activator (e.g., NOBS or TAED); 0% to about 2% carboxymethylcellulose (CMC); about 1% to about 5% polymer (e.g., polycarboxylate or PEG); 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g., optical brightener, fragrance).

[0154] 10) (calculated as acid) about 15% to about 23% linear alkylbenzene sulfonate; about 8% to about 15% alcohol ethoxy sulfate (e.g., C12-15 alcohol, 2-3EO); about 3% to about 9% alcohol ethoxylate (e.g., C12-15 alcohol, 7EO or C12-15 alcohol, 5EO); 0% to about 3% fatty acid (e.g., lauric acid) as soap; about 1% to about 5% aminoethanol; about 5% to An aqueous liquid detergent composition comprising about 10% sodium citrate; about 2% to about 6% hydrotrope (e.g., sodium toluenesulfonate); 0% to about 2% borate; 0% to about 1% carboxymethylcellulose; about 1% to about 3% ethanol; about 2% to about 5% propylene glycol; 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g., polymers, dispersants, fragrances, optical brighteners).

[0155] 11) An aqueous liquid detergent composition comprising (calculated as acid) about 20% to about 32% linear alkylbenzene sulfonate; 6 to 12% alcohol ethoxylate (e.g., C12-15 alcohol, 7EO or C12-15 alcohol, 5EO); about 2% to about 6% aminoethanol; about 8% to about 14% citric acid; about 1% to about 3% borate; 0% to about 3% polymer (e.g., maleic acid / acrylic acid copolymer, anchoring polymer, e.g., lauryl methacrylic acid / acrylic acid copolymer); about 3% to about 8% glycerol; 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 5% minor ingredients (e.g., hydrotropes, dispersants, fragrances, optical brighteners).

[0156] 12) A detergent composition prepared as a granule having a bulk density of at least 600 g / L, comprising about 25% to about 40% anionic surfactants (linear alkylbenzene sulfonates, alkyl sulfonates, α-olefin sulfonates, α-sulfo fatty acid methyl esters, alkanesulfonates, soaps); about 1% to about 10% nonionic surfactants (e.g., alcohol ethoxylates); about 8% to about 25% sodium carbonate; about 5% to about 15% soluble silicates; 0% to about 5% sodium sulfate; about 15% to about 28% zeolite; 0% to about 20% sodium perborate; about 0% to about 5% bleach activator (TAED or NOBS); 0.0001 to 0.1% enzymes (calculated as pure enzyme protein); and 0 to 3% minor ingredients (e.g., fragrances, fluorescent whitening agents).

[0157] 13) A detergent composition according to any one of the above compositions (1) to (12), in which all or a part of the linear alkylbenzene sulfonate is substituted with a (C12 to C18) alkyl sulfate.

[0158] 14) about 9% to about 15% (C12-C18) alkyl sulfate; about 3% to about 6% alcohol ethoxylate; about 1% to about 5% polyhydroxyalkyl fatty acid amide; about 10% to about 20% zeolite; about 10% to about 20% layered disilicate (e.g., SK56 manufactured by Hoechst); about 3% to about 12% sodium carbonate; 0% to about 6% soluble silicate; about 4% to about 8% sodium citrate. from about 13% to about 22% sodium percarbonate; from about 3% to about 8% TAED; from 0% to about 5% polymers (e.g., polycarboxylates and PVP); from 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and from 0 to 5% minor ingredients (e.g., optical brighteners, photobleachers, fragrances, suds suppressors), formulated as granules having a bulk density of at least 600 g / L.

[0159] 15) A detergent composition prepared as a granule having a bulk density of at least 600 g / L, comprising about 4% to about 8% (C12-C18) alkyl sulfate; about 11% to about 15% alcohol ethoxylate; about 1% to about 4% soap; about 35% to about 45% zeolite MAP or zeolite A; about 2% to about 8% sodium carbonate; 0% to about 4% soluble silicate; about 13% to about 22% sodium percarbonate; 1 to 8% TAED; 0% to about 3% carboxymethylcellulose (CMC); 0% to about 3% polymers (e.g., polycarboxylates and PVP); 0.0001 to 0.1% enzyme (calculated as pure enzyme protein); and 0 to 3% minor ingredients (e.g., optical brighteners, phosphonates, fragrances).

[0160] 16) Detergent formulations as described in 1) to 15) above containing stabilized or encapsulated peracid either as an additional ingredient or as a replacement for the bleaching systems already specified.

[0161] 17) Detergent compositions as described above under 1), 3), 7), 9) and 12), wherein perborate is replaced by percarbonate.

[0162] 18) The detergent composition as described above in 1), 3), 7), 9), 12), 14) and 15), further comprising a manganese catalyst, for example one of the compounds described by Hage et al. ((1994) Nature 369:637-639).

[0163] 19) Detergent compositions formulated as non-aqueous detergent solutions containing a liquid nonionic surfactant, such as a linear alkoxylated primary alcohol, a builder system (e.g., phosphates), enzymes and alkali. The detergent may also contain an anionic surfactant and / or a bleaching system.

[0164] As noted above, the amylase polypeptides can be incorporated in concentrations conventionally used in detergents. It is currently contemplated that the enzyme can be added to detergent compositions in an amount equivalent to 0.00001 to 1.0 mg of amylase polypeptide (calculated as pure enzyme protein) per liter of wash liquor.

[0165] The detergent compositions may also contain other conventional detergent ingredients such as deflocculating materials, fillers, suds suppressors, anticorrosive agents, soil suspending agents, sequestrants, soil anti-redeposition agents, drainage agents, dyes, disinfectants, fluorescent agents, thickeners and fragrances.

[0166] The detergent compositions can be formulated as hand (manual) or machine (automatic) laundry detergent compositions comprising a laundry additive composition suitable for pre-treating stained fabrics and a fabric softener composition added to the rinse, or can be formulated as detergent compositions for use in general household hard surface cleaning operations, or can be formulated for hand or automatic dishwashing operations.

[0167] Any of the cleaning compositions described herein may contain any number of additional enzymes. In general, the enzymes must be compatible with the selected detergent (e.g., with respect to pH optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.) and the enzymes must be present in an effective amount. Examples include the following enzymes:

[0168] Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants as well as naturally processed proteins are included. The protease may be a serine protease or a metalloprotease, an alkaline microbial protease, a trypsin-like protease or a chymotrypsin-like protease. Examples of alkaline proteases are subtilisins, particularly proteases from the genus Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (see, for example, WO 89 / 06279). Exemplary proteases also include, but are not limited to, those described in WO 95 / 23221, WO 92 / 21760, WO 95 / 23222, WO 92 / 21760, WO 95 / 23221 ... , Brochure No. 2008010925, Brochure No. 20100566356, Brochure No. 2011072099, Brochure No. 201113022, Brochure No. 2011140364, Brochure No. 2012151534, Brochure No. 2015038792, Brochure No. 2015089441, Brochure No. 2015089447, Brochure No. 2015143360, Brochure No. 201 Brochure No. 6001449, Brochure No. 2016001450, Brochure No. 2016061438, Brochure No. 2016069544, Brochure No. 2016069548, Brochure No. 2016069552, Brochure No. 2016069557, Brochure No. 2016069563, Brochure No. 2016069569, Brochure No. 2016087617, Brochure No. 2016087619 Brochure, US Patent Publication No. 2016145428 Brochure, US Patent Publication No. 2016174234 Brochure, US Patent Publication No. 2016183509 Brochure, US Patent Publication No. 2016202835 Brochure, US Patent Publication No. 2016205755 Brochure, US Patent Application Publication No. 2008 / 0090747, US Patent Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625, Reissue Patent No. 34,No. 606, U.S. Patent Nos. 5,955,340, 5,700,676, 6,312,936, 6,482,628, and 8,530,219; U.S. Provisional Patent Applications Nos. 62 / 331282, 62 / 343618, 62 / 351649, 62 / 437171, 62 / 437174, and 62 / 437509; and PCT application PCT / CN2017 / 0767 49, and metalloproteases described in International Publication Nos. 2007 / 044993, 2009 / 058303, 2009 / 058661, 2014 / 071410, 2014 / 194032, 2014 / 194034, 2014 / 194054 and 2014 / 194117.

[0169] Exemplary commercially available proteases include MAXATASE, MAXACAL, MAXAPEM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX®, EXCELLASE®, PREFERENZ™ Proteases (e.g., P100, P110, P280), EFFECTENZ™ Proteases (e.g., P1000, P1050, P2000), EXCELLENZ™ Proteases (e.g., P1000), ULTIMASE®, and PURAFAST (Danisco US; ALCALASE®, ALCALASE® ULTRA, BLAZE®, BLAZE® EVITY®, BLAZE® EVITY® 16L, CORONASE®, SAVINASE®, SAVINASE® ULTRA, SAVINASE® EVITY®, SAVINASE®, EVERIS®, PRIMASE, DURAZYM, POLARZYME®, OVOZYME®, KANNASE®, LIQUANASE®, EVERIS®, NEUTRASE®, PROGRESS UNO®, RELASE® and ESPERASE® (Novozymes); BLAP™ and BLAP™ variants (Henkel); LAVERGY™ PRO 104L (BASF) and KAP® (B. alkalophilus subtilisin) (Kao). Suitable proteases include naturally occurring proteases or recombinant variants that have been specifically selected or engineered to function at significantly lower temperatures.

[0170] Lipases: Suitable lipases include lipases of bacterial or fungal origin. They include chemically modified, proteolytically modified or recombinant mutants. Examples of useful lipases include lipases from the genus Humicola (synonym Thermomyces), for example from H. lanuginosa (T. lanuginosus) (see, for example, EP 258068 and EP 305216), from H. insolens (see, for example, WO 96 / 13580); Pseudomonas lipases ( For example, from P. alcaligenes or P. pseudoalcaligenes; see, for example, EP 218272), P. cepacia (see, for example, EP 331376), P. stutzeri (see, for example, GB 1,372,034), P. fluorescens, Pseudomonas spp. sp. strain SD705 (see, e.g., WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (see, e.g., WO 96 / 12012); Bacillus lipase (e.g., from B. subtilis; see, e.g., Dartois et al (1993). Biochemica et Biophysica Acta, 1131:253-360), B. stearothermophilus (see, e.g., JP 64 / 744992 A) or B. pumilus (see, e.g., WO 91 / 16422 A).Additional lipase variants contemplated for use in the present preparations include, for example, those described in: WO 92 / 05249, WO 94 / 01541, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, EP 407225 and EP 260105.

[0171] Exemplary commercially available lipases include, but are not limited to, M1 LIPASE, LUMA FAST, and LIPOMAX (Genencor); LIPEX®, LIPOCLEAN®, LIPOLASE®, and LIPOLASE® ULTRA (Novozymes); and LIPASE P (Amano Pharmaceutical Co. Ltd).

[0172] Polyesterases: The compositions may include suitable polyesterases, such as those described in WO 01 / 34899, WO 01 / 14629 and US Pat. No. 6,933,140.

[0173] Amylases: The compositions can be combined with other amylases, including other α-amylases. Such combinations are particularly desirable when different α-amylases exhibit different performance characteristics, resulting in a composition in which the combination of multiple different α-amylases provides the benefits of different α-amylases. Other amylases include, but are not limited to, commercially available amylases such as STAINZYME®, NATALASE®, DURAMYL®, TERMAMYL®, FUNGAMYL®, and BAN™ (Novo Nordisk A / S and Novozymes A / S); RAPIDASE®, POWERASE®, PURASTAR®, and PREFERENZ™ (DuPont Industrial Biosciences). Exemplary α-amylases are disclosed in WO 9418314A1, U.S. Patent Application Publication No. 20080293607, WO 2013063460, WO 10115028, WO 2009061380A2, WO 2014099523, WO 2015077126A1, WO 2013184577, WO 2014164777, WO 9510603, WO 9526397, WO 9623874, WO 9623873, WO 9741213, WO 99194 These are described in Brochure No. 67, Brochure No. 0060060, Brochure No. 0029560, Brochure No. 9923211, Brochure No. 9946399, Brochure No. 0060058, Brochure No. 0060059, Brochure No. 9942567, Brochure No. 0114532, Brochure No. 02092797, Brochure No. 0166712, Brochure No. 0188107, Brochure No. 0196537, Brochure No. 0210355, Brochure No. 2006002643, Brochure No. 2004055178 and Brochure No. 9813481.

[0174] Cellulase: Cellulases may be added to the composition. Suitable cellulases include cellulases of bacterial or fungal origin, including chemically modified or protein modified mutants. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as fungal cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Pat. Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO 89 / 09259. Exemplary cellulases contemplated for use are cellulases that have color care benefits for fabrics. Examples of such cellulases are, for example, those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397 and WO 98 / 08940. Other examples are, for example, the cellulase variants described in WO 94 / 07998; WO 98 / 12307; WO 95 / 24471; PCT / DK98 / 00299; EP 531 315; U.S. Pat. Nos. 5,457,046; 5,686,593; and 5,763,254. Exemplary cellulases include those described in WO 2005054475, WO 2005056787, U.S. Pat. Nos. 7,449,318, 7,833,773, and 4,435,307; EP 0495257; and U.S. Provisional Patent Applications 62 / 296,678 and 62 / 435340.Exemplary commercially available cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®, CAREZYME®, CAREZYME® PREMIUM, ENDOLASE®, and RENOZYME® (Novozymes); REVITALENZ® 100, REVITALENZ® 200 / 220, and REVITALENZ® 2000 (Danisco US); and KAC-500(B) (Kao Corporation).

[0175] Mannanases: Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO 2016007929; U.S. Patent Nos. 6,566,114, 6,602,842, and 6,440,991; and International Applications PCT / US2016 / 060850 and PCT / US2016 / 060844. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO 2016007929; U.S. Patent Nos. 6,566,114, 6,602,842, and 6,440,991; and International Applications PCT / US2016 / 060850 and PCT / US2016 / 060844.

[0176] Peroxidase / Oxidase: Suitable peroxidase / oxidase contemplated for use in the present composition include those of plant, bacterial or fungal origin. Chemically modified mutants or protein modified mutants are included. Examples of useful peroxidases include peroxidases from the genus Coprinus, for example C. cinereus, as described in WO 93 / 24618, WO 95 / 10602 and WO 98 / 15257, and variants thereof. Commercially available peroxidases include, for example, GUARDZYME™ (available from Novo Nordisk A / S and Novozymes A / S).

[0177] The detergent compositions may also include 2,6-β-D-fructan hydrolase, which is effective for removing / cleaning biofilms present on domestic and / or industrial fabrics / laundry.

[0178] The detergent enzymes can be included in the detergent composition by adding a separate additive containing one or more enzymes, or by adding a combined additive containing all of the enzymes. The detergent additive, i.e., separate additive or combined additive, can be prepared, for example, as granules, liquids, slurries, etc. Typical detergent additive preparations include, but are not limited to, granules, especially non-powdered granules, liquids, especially stabilized liquids or slurries.

[0179] Non-dusted granules can be prepared, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and can be optionally coated by methods known in the art. Examples of waxy coating substances are poly(ethylene oxide) products (e.g., polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and furthermore 15 to 80 ethylene oxide units are present; fatty alcohols; fatty acids; mono- and di- and triglycerides of fatty acids. Examples of film-forming coating substances suitable for application by the fluidized bed technique are described, for example, in GB 1 483 591. Liquid enzyme preparations can be stabilized, for example, by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid according to established methods. The protected enzymes can be prepared according to the method disclosed in EP 238,216.

[0180] The detergent composition may be in any convenient form, for example, a bar, tablet, powder, granule, paste or liquid. Liquid detergents may typically be aqueous, containing up to about 70% water and 0% to about 30% organic solvent. Compact detergent gels containing up to about 30% water are also contemplated. The detergent composition may optionally include one or more surfactants, which may be semi-polar and / or anionic and / or cationic and / or non-ionic, including zwitterionic. The surfactant may be present within a broad range of about 0.1% to about 60% by weight.

[0181] If included therein, the detergent will typically contain from about 1% to about 40% of an anionic surfactant such as, for example, linear alkylbenzene sulfonates, alpha-olefin sulfonates, alkyl sulfates (fatty alcohol sulfates), alcohol ethoxy sulfates, secondary alkane sulfonates, alpha-sulfo fatty acid methyl esters, alkyl or alkenyl succinic acids or soaps.

[0182] If included therein, the detergent will typically contain from about 0.2% to about 40% of a nonionic surfactant such as, for example, alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethylamine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides, polyhydroxyalkyl fatty acid amides, or N-acyl-N-alkyl derivatives of glucosamine ("glucamides").

[0183] The detergent may contain from 0% to about 65% of detergent builders or complexing agents, such as, for example, zeolites, diphosphates, triphosphates, phosphonates, carbonates, citrates, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, alkyl and alkenyl succinic acids, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

[0184] The detergent may include one or more polymers. Exemplary polymers include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers, and methacrylic acid / lauryl acrylate copolymers.

[0185] The enzymes of the detergent compositions can be stabilized using conventional stabilizers, such as polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives (e.g., aromatic boric acid esters) or phenylboronic acid derivatives (e.g., 4-formylphenylboronic acid). The compositions can be prepared, for example, as described in WO 92 / 19709 and WO 92 / 19708.

[0186] In the present detergent composition, it is specifically contemplated that the enzyme variant may be added in an amount equivalent to about 0.01 to about 100 mg of enzyme protein per liter of wash solution (e.g., about 0.05 to about 5.0 mg of enzyme protein per liter of wash solution or 0.1 to about 1.0 mg of enzyme protein per liter of wash solution).

[0187] A number of exemplary detergent formulations to which the amylase can be added (or in some cases has been identified as a component thereof) are described in WO2013063460. These include commercially available unit dose detergent formulations / packages such as PUREX® UltraPacks (Henkel), FINISH® Quantum (Reckitt Benckiser), CLOROX™ 2 Packs (Clorox), OxiClean Max Force Power Paks (Church & Dwight), TIDE® Stain Release, CASCADE® ActionPacs and TIDE® Pods (Procter & Gamble), PS.

[0188] 7.6. Methods for assessing amylase activity in detergent compositions Many α-amylase cleaning assays are known in the art, including swatch and microswatch assays, and the accompanying examples describe only a few such assays.

[0189] In order to more fully illustrate the present compositions and methods and their advantages, the following specific examples are given, with the understanding that they are illustrative rather than limiting.

[0190] 8. Brewing composition The variant amylases can be components of brewing compositions used in the brewing process, i.e., in producing fermented malt beverages. Non-fermentable carbohydrates form the majority of the dissolved solids in the final beer. This residue remains due to the inability of malt amylase to hydrolyze the α-1,6-linkages in starch. Non-fermentable carbohydrates contribute approximately 50 calories per 12 ounces of beer. Amylases, in combination with glucoamylase and optionally pullulanase and / or isoamylase, convert starch to dextrins and fermentable sugars, helping to reduce the residual non-fermentable carbohydrates in the final beer.

[0191] 9. Reduction of iodine positive starch Mutant amylases, when used in liquefaction and / or saccharification processes, can reduce iodine positive starch (IPS). One source of IPS is amylose and / or retrograded starch polymers that escape hydrolysis. Retrogradation occurs naturally in starch pastes or gels undergoing retrogradation due to the tendency of starch molecules to bond with each other followed by an increase in crystallinity. Low-concentration solutions become increasingly turbid due to the progressive association of starch molecules into larger bodies. Spontaneous precipitation occurs and the precipitated starch appears to show a tendency to revert to its initial state of cold water insolubility. Higher concentration pastes after cooling and setting into gels steadily become firmer due to the increased association of starch molecules upon retrogradation. This occurs due to a strong tendency for hydrogen bonds to form between hydroxyl groups on adjacent starch molecules. See J. A. Radley, ed., Starch and its Derivatives 194-201, Chapman and Hall, London (1968). The use of the variant amylase is expected to improve overall process performance by reducing the amount of IPS.

[0192] All references mentioned herein are incorporated by reference in their entirety for all purposes. In order to further illustrate the present compositions and methods and their advantages, the following specific examples are provided with the understanding that they are illustrative rather than limiting. EXAMPLES

[0193] Generation of BspAmy24 mutants Proteins were prepared as follows: DNA sequences encoding the proteins of interest, signal peptides for secretion and additional 5' and 3' sequences for amplification and subcloning were ordered from commercial suppliers. Standard procedures were used to insert these DNA sequences into bacterial vectors for the expression of secreted proteins in Bacillus subtilis cells. Constructs were verified by DNA casing. Cells were grown for 68 hours in expression medium suitable for the expression of secreted proteins from B. subtilis. Cells were separated from the protein-containing supernatant by centrifugation followed by filtration through a 0.45 μm membrane (EMD Millipore). Further purification was achieved through ion exchange chromatography using Phenyl Sepharose 6 Fast Flow resin (GE Healthcare). Protein concentrations were determined by high performance liquid chromatography (HPLC) and absorbance at 280 nm. The expressed and purified variants of BspAmy24 (SEQ ID NO: 1) are shown in Table 2. [Table 2]

[0194] The starch cleaning activity of the mutants was evaluated using rice starch on a commercial cotton test fabric (CS28, Center for Test Materials, Netherlands) cut into 5.5 mm circular swatches. Two swatches were placed in each well of a 96-well Corning 9017 flat-bottom polystyrene microtiter plate. A protein solution from a commercial automatic dishwashing detergent formulation containing inactivated enzymes was added to the test fabric. The solution was shaken for 15 min at 50°C. A sample of the supernatant was evaluated for dye release by measuring the absorbance of the solution at 488 nm using a spectrophotometer.

[0195] Commercial automatic dishwasher detergent formulations containing inactivated enzymes were prepared as follows: One individually wrapped packet of CASCADE® PLATINUM™ or FINISH® QUANTUM™ dishwasher detergent formulation was placed in 1 L of distilled water and stirred to dissolve the packet. The solution was incubated at 85-90°C for 10 hours. After cooling, the volume of the solution was increased to the approximate volume of a North American dishwasher (3.3 L) with distilled water and the water hardness was increased to 150 PPM (8.76 GPG) with a calcium to magnesium ratio of 3:1. The results of the cleaning assay are shown in Figures 1-4. The BspAmy24 variant performed better compared to STAINZYME® (Novozymes), a well-known detergent amylase.

[0196] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety into this specification for all purposes and to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A mutant α-amylase, The amino acid sequence of said mutant α-amylase comprises a combination of a deletion of two amino acid residues corresponding to R181 and G182 in SEQ ID NO:1, and a substitution of three amino acid residues corresponding to Q172R, A186G, and I324M, said mutant α-amylase having at least 91% amino acid sequence identity to SEQ ID NO:1 used for numbering purposes, and said mutant α-amylase having improved cleaning performance in automatic dishwashing compared to a reference α-amylase which differs from the mutant α-amylase only by the absence of said deletion and substitution mutations.

2. A polynucleotide encoding the mutant α-amylase according to claim 1, an expression vector comprising said polynucleotide, or an expression host cell comprising said polynucleotide or said expression vector.

3. 13. A composition for liquefying starch comprising the mutant α-amylase of claim 1.

4. A detergent composition comprising the mutant α-amylase of claim 1.

5. 13. A method for converting starch to oligosaccharides comprising the step of contacting starch with an effective amount of the mutant α-amylase of claim 1.

6. 13. A method for removing starchy stains or soils from a surface, comprising the steps of contacting the surface with an effective amount of the mutant α-amylase of claim 1 and allowing the mutant α-amylase to hydrolyze starch components present in the starchy stains to produce smaller starch derived molecules that are soluble in an aqueous composition, thereby removing the starchy stains from the surface.

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