α-amylase that produces maltopentose / maltohexasose variants

Abstract

This invention discloses compositions and methods relating to maltopentose / maltohexasose α-amylases. Variant α-amylases can be used, for example, for starch liquefaction and saccharification, for cleaning starch stains in clothing, dishwashing, and other applications, for textile processing (e.g., desizing), for improving digestibility in animal feed, and for baking and brewing.

Description

Technical Field

[0001] This invention discloses compositions and methods relating to maltopentose / maltohexasose variant α-amylases. These variant α-amylases can be used, for example, for cleaning starch stains, starch liquefaction and saccharification, textile desizing, baking, and brewing. Background Technology

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

[0003] Alpha-amylases hydrolyze starch, glycogen, and related polysaccharides by randomly cleaving internal α-1,4-glycosidic bonds. Alpha-amylases, particularly those from the genus *Bacilli*, have been used for a wide variety of purposes, including starch liquefaction and saccharification, textile desizing, starch modification in the paper and pulp industry, brewing, baking, syrup production for the food industry, feedstock production for fermentation processes, and increasing digestibility in animal feed. These enzymes are also used to remove starchy dirt and stains during dishwashing and laundry.

[0004] The products of α-amylase hydrolysis of starch vary in the number of consecutive glucose molecules. Most commercial α-amylases produce a range of products from glucose (G1) to maltoheptaose (G7). For reasons not fully understood, α-amylases that produce large amounts of maltopentose and maltohexaose appear to be particularly useful for certain commercial applications, including incorporation into detergent cleaning compositions. Numerous publications have described α-amylases that produce maltopentose / maltohexaose, among others. Nevertheless, the need for more robust and higher-performing engineered α-amylase molecules remains. Summary of the Invention

[0005] The compositions and methods of the present invention relate to maltopentose / maltohexasose variant amylase polypeptides and methods of using them. Aspects and examples of the compositions and methods of the present invention are summarized in the following numbered paragraphs:

[0006] 1. In one aspect, a recombinant, non-naturally occurring parental variant α-amylase molecule is provided, comprising a mutation at position 91 (numbered with reference to SEQ ID NO:1) and a mutation at an amino acid residue at the bottom of the α-amylase TIM tube structure, said bottom amino acid residues being defined as residues 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325, 362, 363, and 364, wherein the wild-type amino acid residue present at position 28 of said parental molecule is positively charged.

[0007] 2. In some embodiments of the variant α-amylase as described in paragraph 1, the mutation at position 91 is the replacement of a naturally occurring residue with a positively charged residue.

[0008] 3. In some embodiments of the variant α-amylase as described in paragraph 1 or 2, the mutation at position 91 is the substitution of a naturally occurring residue with a spermine amino group (i.e., X91R).

[0009] 4. In some embodiments of the variant α-amylase as described in any one of paragraphs 1-3, at least one mutation at the bottom of the α-amylase TIM tube structure is selected from the group consisting of: X40N, X40D, X100F, X100L, X263Y, X288D, X288K, X288Q, X324R, X324N, X324M, X364L, and X364M.

[0010] 5. In some embodiments of the variant α-amylase as described in any one of paragraphs 1-4, at least one mutation at the bottom of the α-amylase TIM tube structure is selected from the group consisting of: T40N, T40D, Y100F, Y100L, F263Y, S288D, S288K, S288Q, I324R, I324N, I324M, Y364L, and Y364M.

[0011] 6. In another aspect, a recombinant, non-naturally occurring variant of α-amylase is provided, comprising arginine at position 91 and at least one of the following features not present in naturally occurring α-amylase: N or D at position 40, F or L at position 100, Y at position 263, D, K or Q at position 288, R, N or M at position 324, or L or M at position 364.

[0012] 7. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-6 further comprises a mutation at residues in a loop containing surface-exposed residues 167, 169, 171, 172, and 176, numbered with reference to SEQ ID NO:1.

[0013] 8. In some embodiments of the variant α-amylase as described in paragraph 7, at least one mutation in the loop is selected from the group consisting of: X167F, X169H, X171Y, X172R, X172N, and X176S.

[0014] 9. In some embodiments of the variant α-amylase as described in paragraph 8, at least one mutation in the loop is selected from the group consisting of: W167F, Q169H, R171Y, Q172R, Q172N, and R176S.

[0015] 10. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-6 further comprises F at position 167, H at position 169, Y at position 171, R or N at position 172, or S at position 176, as numbered with reference to SEQ ID NO:1.

[0016] 11. In another aspect, a recombinant, non-naturally occurring variant of α-amylase is provided, comprising a mutation at position 172 and a mutation at position 288, as indicated by reference to SEQ ID NO:1.

[0017] 12. In another aspect, a recombinant, non-naturally occurring variant of α-amylase is provided, comprising arginine or asparagine at position 172 and aspartic acid at position 288, as indicated by reference to SEQ ID NO:1.

[0018] 13. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-12 further comprises a mutation at position 116 and / or 281, as indicated by reference to SEQ ID NO:1.

[0019] 14. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-12 further comprises arginine at position 116 or serine at position 281, as indicated by reference to SEQ ID NO:1.

[0020] 15. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-14 further comprises a mutation at position 190 and / or 244, as indicated by reference to SEQ ID NO:1.

[0021] 16. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-14 has proline at position 190 and / or alanine, glutamic acid or glutamine at position 244, as numbered with reference to SEQ ID NO:1.

[0022] 17. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-16 further comprises the deletion of at least two residues of SEQ ID NO:1, corresponding to R181, G182, T183, and G184.

[0023] 18. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-16 further comprises paired deletions of residues corresponding to R181 and G182 or residues T183 and G184.

[0024] 19. In another aspect, a recombinant, non-naturally occurring variant of α-amylase is provided, comprising:

[0025] (i) selected using SEQ ID NO:1 as the substitute for the group consisting of:

[0026] (a)X40N-X91R-X169H-X183M-X281N,

[0027] (b)X172R-X190P-X288D

[0028] (c)X172R-X244E-X288D-X474R、

[0029] (d)X91R-X172R-X190P-X324M、

[0030] (e)X40N-X91R-X190P-X263Y、

[0031] (f)X40N-X91R-X244E-X364L、

[0032] (g)X91R-X172R-X190P-X324R、

[0033] (h)X91R-X116R-X172R-X244E-X281S-X288D,

[0034] (i)X40N-X91R-X100F-X116R-X172N-X244Q-X281S,

[0035] (j)X40N-X91R-X172R-X244Q-X263Y-X281S,

[0036] (k)X91R-X172R-X190P-X324N、

[0037] (l)X40D-X91R-X172R-X190P-X281S-X324R, and

[0038] (m)X364L; and

[0039] (ii) Paired deletion of residues, wherein the residues are selected from the group consisting of:

[0040] 181 and 182, and

[0041] 183 and 184.

[0042] 20. In some embodiments, the variant α-amylase as described in paragraph 19 comprises:

[0043] (i) selected using SEQ ID NO:1 as the substitute for the group consisting of:

[0044] (a)T40N-S91R-Q169H-T183M-H281N,

[0045] (b)Q172R-E190P-S288D

[0046] (c)Q172R-S244E-S288D-S474R、

[0047] (d)S91R-Q172R-E190P-I324M、

[0048] (e)T40N-S91R-E190P-F263Y、

[0049] (f)T40N-S91R-S244E-Y364L、

[0050] (g)S91R-Q172R-E190P-I324R、

[0051] (h)S91R-W116R-Q172R-S244E-H281S-S288D,

[0052] (i)T40N-S91R-Y100F-W116R-Q172N-S244Q-H281S,

[0053] (j)T40N-S91R-Q172R-S244Q-F263Y-H281S、

[0054] (k)S91R-Q172R-E190P-I324N、

[0055] (l)T40D-S91R-Q172R-E190P-H281S-I324R, and

[0056] (m)Y364L; and

[0057] (ii) Paired deletion of residues, wherein the residues are selected from the group consisting of:

[0058] R181 and G182, and

[0059] T183 and G184.

[0060] 21. In another aspect, a recombinant, non-naturally occurring variant of α-amylase is provided, comprising three or more of the following features: (a) D or N at position 40 and / or R at position 91, and (b) F at position 100, Y at position 263, D at position 288, M, N or R at position 324, and / or L at position 364, optionally in combination with (c) H at position 169, M at position 183M, N or S at position 281, N or R at position 172, P at position 190, E, Q or R at position 244, R at position 474, and / or R at position 116, and (d) optionally in combination with (e) paired deletions at positions 181 and 182 or 183 and 184, in all cases numbered using SEQ ID NO:1.

[0061] 22. In some embodiments, the variant α-amylase as described in any one of paragraphs 1-21 has at least 70%, at least 80%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0062] 23. In another aspect, a detergent composition is provided, the detergent composition comprising a variant α-amylase as described in any one of paragraphs 1-22.

[0063] 24. In some embodiments, the detergent composition as described in paragraph 23 further comprises a variant of Bacillus gibsonii protease having amino acid substitutions X39E, X99R, X126A, X127E, and X128G, and further comprises reference SEQ ID For NO:5, select one or more additional substitutions from the following groups: N74D-M211L-N253P, R179Q-M211L-N253P, N74D-N253P, N85R-G160Q-R179Q-M211L-N212S-N253P, R179Q-N253P, G160Q-R179Q-M211L-N212S-N253P, R179Q-M211L, G160Q-R179Q-M211L-N253P, G160Q-R179Q-N212S-N253P N74D-M211L, M211L-N242D, G160Q-R179Q-M211L-N212S, N74D-R179Q-M211L-N253P, G160Q-R179Q-M211L, G160Q-R179Q-N253P, N 74D-Q200L-M211L, N74D-G160Q-N212S-N253P, N74D-G160Q-M211L-N253P, G160Q-R179Q, G160Q-R179Q-N212S, N74D-G160Q-N253 P. N74D-G160Q-R179Q-M211L-N212S-N253P, N74D-N085R-G160Q-R179Q-M211L, N74D-G160Q-M211L-N212S-N253P, N74D-N085R- N116R-Q200L-Q256E, N74D-G160Q-R179Q-N212S-N253P, N74D-G160Q-M211L-N212S, N74D-G160Q, N74D-G160Q-R179Q-M211L-N25 3P, N74D-R179Q-M211L, N74D-G160Q-N212S, N74D-G160Q-M211L, N74D-G160Q-R179Q-N253P, N74D, N74D-G160Q-R179Q-M211L-N2 12S, N74D-N085R-M211L-N212S, N74D-G160Q-R179Q-N212S, N74D-G160Q-R179Q-M211L, N74D-M211L-Q256E, N74D-G160Q-R179Q,R179Q-M211L-N212S-N253P, R179Q-M211L-N212S, N74D-N085R-R179Q-M211L-N212S, N74D-M211L-N212S, N74D-R179Q-M211L-N212 S, N74D-M211L-N242D, N74D-Q200L-M211L-Q256E, N74D-Q200L-M211L-N242D-Q256E, N74D-Q200L, N74D-M211N-N212Q, N74D-M211N- N212Q-Q256E, N74D-M211N-Q256E, N74D-M211Q, N74D-M211Q-N212Q, N74D-M211Q-N212Q-Q256E, N74D-M211Q-Q256E, N74D-N198A-M2 11Q, N74D-N198A-M211Q-N212Q, N74D-N198A-M211Q-Q256E, N74D-N198G-M211Q, N74D-N198G-M211Q-N212Q, N74D-N198G-M211Q-Q25 6E, N74D-N198K-M211Q-N212Q, N74D-N198L-M211Q-N212Q, N74D-N198Q-M211Q-N212Q, N74D-N198R-M211Q-N212Q, N74D-N198T-M21 1Q-N212Q, N74D-N198V-M211Q-N212Q, N74D-N212Q-Q256E, N74D-Q256E, N74D-R207Q, N74D-R207Q-M211N, N74D-R207Q-M211N-N212Q The sequences N74D-R207Q-M211N-N212Q-Q256E, N74D-R207Q-M211N-Q256E, N74D-R207Q-M211Q, N74D-R207Q-M211Q-N212Q, N74D-R207Q-M211Q-N212Q-Q256E, N74D-R207Q-N212Q, N74D-R207Q-N212Q-Q256E, N74D-R207Q-Q256E, N74D-N198S-M211Q, and N74D-N198L-M211Q, and have at least 90% amino acid sequence identity with SEQ ID NO:6.

[0064] 25. In another aspect, a method for converting starch into oligosaccharides is provided, the method comprising contacting the starch with an effective amount of a variant α-amylase as described in any one of paragraphs 1-22.

[0065] 26. In another aspect, a method for removing starch stains or dirt from a surface is provided, the method comprising contacting the surface with an effective amount of a variant α-amylase as described in any one of paragraphs 1-22, and allowing the polypeptide to hydrolyze the starch component present in the starch stain to produce smaller starch-derived molecules dissolved in an aqueous composition, thereby removing the starch stain from the surface.

[0066] 27. On the other hand, nucleic acids are provided that encode variant α-amylases as described in any one of paragraphs 1-22.

[0067] 28. On the other hand, a host cell is provided, which contains nucleic acids as described in paragraph 27.

[0068] These and other aspects and embodiments of the compositions and methods of the present invention will become clear from the following description and the accompanying examples. Attached Figure Description

[0069] Figure 1 The Clustal W amino acid sequence alignments for AA2560, AA707, AA560, and AAI10 are shown.

[0070] Figure 2 This shows a view of AA2560 amylase through the central β-tube. The residues at the bottom of the tube are shown as spherical α-carbon positions. The amino acid numbers at these positions are given.

[0071] Figure 3 This shows a side view of the central β-tube of AA2560 amylase, which is helically oriented at amino acids 82-94. The residues at the bottom of the tube are shown as spherical α-carbon positions and indicated position numbers.

[0072] Figure 4 This is a cross-sectional view showing two spirals in the AA2560 structural model. Positions 28 and 91 are indicated by rods. Figure 4 Image A shows wild-type Arg28 and Ser91 represented in rod form. Figure 4 Image B shows the close proximity of two Arg residues in the S91R variant, represented in a rod shape.

[0073] Figure 5 This is a table showing the performance of the combined variants AA2560, AA560, AA707, and AAI10 in cleaning assays. Detailed Implementation

[0074] Compositions and methods relating to maltopentose / maltohexasose variant amylases are described. The variants are discovered through experimental methods detailed in the accompanying examples. Exemplary applications of the variant amylases include cleaning starch stains in dishwashing, clothing, and other applications; starch liquefaction or saccharification; textile processing (e.g., desizing); improving digestibility in animal feed; and baking and brewing. These and other aspects of these compositions and methods are described in detail below.

[0075] Before describing the various aspects and embodiments of the compositions and methods of the present invention, the following definitions and abbreviations are described.

[0076] 1. Definitions and Abbreviations

[0077] Based on this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms “a / an” and “the” include a plural indicator unless the context clearly indicates otherwise. Thus, for example, reference to “enzyme” includes multiple such enzymes, and reference to “dosage” includes reference to a single or multiple doses known to those skilled in the art and their equivalents.

[0078] This document is organized into several parts for ease of reading; however, the reader will understand that statements made in one part may apply to other parts. In this way, the headings used for different parts of this disclosure should not be construed as limiting.

[0079] 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. For clarity, the following terms are defined as follows.

[0080] 1.1. Abbreviations and Acronyms

[0081] Unless otherwise stated, the following abbreviations / acronyms have the following meanings:

[0082] ℃ Celsius

[0083] ADW Automatic Dishwasher

[0084] dH2O or DI deionized water

[0085] dIH2O deionized water, Milli-Q filtration

[0086] DNA deoxyribonucleic acid

[0087] EC Enzyme Committee

[0088] g or gm grams

[0089] GA glucosyl amylase

[0090] H2O water

[0091] HDD Heavy-Duty Powder Detergent

[0092] HDL High Density Liquid Detergent

[0093] hr hours

[0094] HSG High-Foaming Granular Detergent

[0095] kDa (kilodaltons)

[0096] kg

[0097] M Moore

[0098] mg

[0099] min minutes

[0100] mL and ml

[0101] mm

[0102] mM millimole

[0103] MW (Molecular Weight)

[0104] MWU Modified Wohlgemuth unit; 1.6 x 10 -5 mg / MWU = Active Unit

[0105] PI performance index

[0106] ppm (parts per million), for example, μg protein / g dry solids.

[0107] sec seconds

[0108] species

[0109] U unit

[0110] v / v volume / volume

[0111] w / v weight / volume

[0112] w / w weight / weight

[0113] wt% (weight percentage)

[0114] μg micrograms

[0115] μL and μl

[0116] μm micrometer

[0117] μM micromolar

[0118] 1.2. Definition

[0119] The term "α-amylase" or "starch-degrading enzyme," or simply amylase, refers to an enzyme that, among other things, catalyzes the degradation of starch. α-Amylase is a hydrolytic enzyme that cleaves the α-D-(1→4)O-glycosidic bonds in starch. Typically, α-amylase (EC 3.2.1.1; α-D-(1→4)-glucan-glucan hydrolase) is defined as an endoglucanase that randomly cleaves the α-D-(1→4)O-glycosidic bonds within the starch molecule, producing a polysaccharide containing three or more (1-4)-α-linked D-glucose units. Conversely, exoglucanases, such as β-amylase (EC 3.2.1.2; α-D-(1→4)-glucan-maltose hydrolase) and some product-specific α-amylases (such as maltose α-amylase (EC 3.2.1.133)), cleave the polysaccharide molecule from the non-reducing end of the substrate. β-amylases, α-glucosidases (EC 3.2.1.20; α-D-glucosidase), glucosylamylases (EC 3.2.1.3; α-D-(1→4)-glucanase), and product-specific amylases (such as maltodextrinase (EC 3.2.1.60) and maltohexasylase (EC 3.2.1.98)) can produce maltooligosaccharides of specific lengths or specific maltooligosaccharides rich in syrup. Some bacterial α-amylases primarily produce maltodextrin (G4), maltopentose (G5), or maltohexasose (G6) from starch and associated α-1,4-glucan, while most α-amylases further convert them into glucose and / or maltose as the final product. G6 amylases (such as those derived from Bacillus sp. DSM 12649, i.e. STAINZYME) TM The AA560 amylase (of the parent) and Bacillus species 707 amylase, also known as maltohexasaccharide α-amylase (EC 3.2.1.98), are technically exonucleases, but have a similar structure to α-amylases and appear to respond to some of the same beneficial mutations in certain cases.

[0120] In this article, "enzyme unit" refers to the amount of product formed per assay under specific conditions. For example, "glucosylamylase activity unit" (GAU) is defined as the amount of enzyme that produces 1 g of glucose per hour from a soluble starch substrate (4% DS) at 60°C and pH 4.2. "Soluble starch unit" (SSU) is the amount of enzyme that produces 1 mg of glucose per minute from a soluble starch substrate (4% DS) at pH 4.5 and 50°C. DS refers to "dry solids".

[0121] The term "starch" refers to any material composed of complex polysaccharide carbohydrates from plants, which are composed of carbohydrates with the formula (C6H12H2O)10 O5) x (Where "X" can be any integer) consists of amylose and amylopectin. The term includes plant-based materials such as cereals, grains, grasses, tubers, and roots, and more particularly, materials obtained from wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, milo, potato, sweet potato, and tapioca starch. The term "starch" includes granular starch. The term "granular starch" refers to raw starch (i.e., uncooked starch), for example, starch that has not undergone gelatinization.

[0122] As used herein, the term "liquefaction" or "liquidation" refers to the process of converting starch into dextrins with lower viscosity and shorter chain lengths.

[0123] Regarding polypeptides, the terms "wild-type," "parent," or "reference" refer to naturally occurring polypeptides that do not contain artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, regarding polynucleotides, the terms "wild-type," "parent," or "reference" refer to naturally occurring polynucleotides that do not include artificial nucleoside changes. However, note that polynucleotides encoding wild-type, parent, or reference polypeptides are not limited to naturally occurring polynucleotides and encompass any polynucleotide encoding wild-type, parent, or reference polypeptides.

[0124] The reference to wild-type polypeptides should be understood to include the mature form of the polypeptide. A "mature" polypeptide or its variants are polypeptides or variants in which the signal sequence is absent, for example, polypeptides that have never been cleaved in their mature form during or after polypeptide expression.

[0125] The term "variant" for polypeptides refers to a polypeptide that differs from a specified wild-type, parent, or reference polypeptide because it includes one or more naturally occurring or artificially created amino acid substitutions, insertions, or deletions. Similarly, the term "variant" for polynucleotides refers to a polynucleotide that differs from a specified wild-type, parent, or reference polynucleotide in terms of its nucleotide sequence. The characteristics of the wild-type, parent, or reference polypeptide or polynucleotide will be apparent from the context.

[0126] In the case of the α-amylase of the present invention, "activity" refers to α-amylase activity, which can be measured as described herein.

[0127] The term "performance benefit" refers to an improvement in the desired properties of a molecule. Exemplary performance benefits include, but are not limited to: increased starch substrate hydrolysis; enhanced liquefaction properties of cereal, cereal, or other starch substrates; enhanced cleaning properties; enhanced thermal stability; enhanced detergent stability; enhanced storage stability; increased solubility; altered pH profile; decreased calcium dependence; increased specific activity; substrate specificity modification; substrate binding modification; pH-dependent activity modification; pH-dependent stability modification; increased oxidative stability; and increased expression. In some cases, performance benefits are achieved at relatively low temperatures. In other cases, performance benefits are achieved at relatively high temperatures.

[0128] The terms "protease" and "proteinase" refer to enzyme proteins that have the ability to perform "protein hydrolysis" or "protein hydrolysis cleavage," which means hydrolyzing the peptide bonds that link the amino acids together in the peptide or polypeptide chain that forms the protein. This activity of a protease as a protein-digesting enzyme is called "proteolytic activity."

[0129] The term "serine protease" refers to enzymes that cleave peptide bonds in proteins, where the serine residue acts as a nucleophilic amino acid at the enzyme's active site. Based on their structure, serine proteases are divided into two main categories: chymotrypsin-like (trypsin-like) and subtilisin-like. Serine proteases, especially subtilisin, are most commonly used in laundry and dishwashing detergents.

[0130] "Combined variants" are variants that include two or more mutations, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, deletions, and / or insertions.

[0131] The term "recombinant" when used to refer to subject cells, nucleic acids, proteins, or vectors indicates that the subject has been modified from its natural state. Thus, for example, recombinant cells express genes not found in their natural (non-recombinant) form, or express natural genes at levels different from those found in nature, or under conditions different from those found in nature. Recombinant nucleic acids differ from their natural sequence by one or more nucleotides and / or are operatively linked to a heterologous sequence, such as a heterologous promoter in an expression vector. Recombinant proteins may differ from their natural sequence by one or more amino acids and / or be fused to a heterologous sequence. A vector containing a nucleic acid encoding an amylase is a recombinant vector.

[0132] The terms “recycled,” “isolated,” and “separate” refer to compounds, proteins (peptides), cells, nucleic acids, amino acids, or other specified materials or components removed from at least one other material or component naturally present in or associated with them. “Isolated” peptides include, but are not limited to, culture media containing secreted peptides expressed in heterologous host cells.

[0133] The term "purified" refers to material in a relatively pure state (e.g., isolated polypeptides or polynucleotides), for example, at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

[0134] The term "enriched" refers to material that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure (e.g., isolated polypeptides or polynucleotides).

[0135] The terms "thermally stable" and "thermally stable" for enzymes refer to the ability of an enzyme to retain its activity after exposure to elevated temperatures. The thermostability of an enzyme (such as amylase) is measured by its half-life (t1 / 2), given in minutes, hours, or days, during which half of the enzyme's activity is lost under defined conditions. The half-life can be calculated by measuring the residual α-amylase activity after exposure to (i.e., challenged to) elevated temperatures.

[0136] The "pH range" of an enzyme refers to the range of pH values ​​at which the enzyme exhibits catalytic activity.

[0137] The terms “pH stable” and “pH stability” for enzymes refer to the ability of an enzyme to maintain its activity for a predetermined period of time (e.g., 15 min, 30 min, 1 hour) over a wide range of pH values.

[0138] The term "amino acid sequence" is synonymous with and used interchangeably with the terms "polypeptide," "protein," and "peptide." When such amino acid sequences exhibit activity, they can be called "enzymes." Amino acid sequences are represented using standard N-terminal to C-terminal orientation (i.e., N→C) using conventional single-letter or three-letter codes for amino acid residues.

[0139] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and may contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention cover nucleotide sequences encoding specific amino acid sequences. Unless otherwise stated, nucleic acid sequences are presented in a 5′ to -3′ orientation.

[0140] "Synthetic" molecules are synthesized in vitro through chemical or enzymatic processes rather than being produced by living organisms.

[0141] In the context of inserting nucleic acid sequences into cells, the term “introduction” means “transfection,” “conversion,” or “transduction” as known in the art.

[0142] A “host strain” or “host cell” is an organism in which an expression vector, bacteriophage, virus, or other DNA construct has been introduced, including a polynucleotide encoding a target polypeptide (e.g., amylase). Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the target polypeptide and / or fermenting sugars. The term “host cell” includes protoplasts derived from cells.

[0143] The term "heterogeneous" in relation to polynucleotides or proteins refers to polynucleotides or proteins that are not naturally present in host cells.

[0144] The term "endogenous" in relation to polynucleotides or proteins refers to polynucleotides or proteins that are naturally present in host cells.

[0145] The term "expression" refers to the process of producing polypeptides based on nucleic acid sequences. This process includes both transcription and translation.

[0146] A "signal sequence" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates its secretion outside the cell. Mature forms of extracellular proteins lack the signal sequence that is cleaved during secretion.

[0147] "Biologically active" refers to a sequence that has a specified biological activity, such as enzyme activity.

[0148] The term "specific activity" refers to the number of moles of substrate that can be converted into a product per unit time by an enzyme or enzyme preparation under specific conditions. Specific activity is usually expressed as units (U) / mg protein.

[0149] As used in this article, “water hardness” is a measure of the minerals (e.g., calcium and magnesium) present in water.

[0150] "Cultured cell material containing amylase" or similar terms refer to cell lysates or supernatants (including media) that include amylase as a component. Cell material can be derived from a heterologous host and is grown in a culture with the aim of producing amylase.

[0151] "Sequence identity percentage" refers to the percentage of amino acid residues in a given sequence that are identical to those in a specified 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:

[0152]

[0153] Deletions are considered as different residues compared to the reference sequence.

[0154] The term "dry solids content" (ds) refers to the total solids of a slurry as a percentage of dry weight. The term "slurry" refers to an aqueous mixture containing insoluble solids.

[0155] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a biochemical production process in which microorganisms, such as ethanol-producing microorganisms, and at least one enzyme, such as amylase, are present in the same process step. SSF includes the simultaneous hydrolysis of a starch substrate (particulate, liquefied, or dissolved) into sugars (including glucose) and the fermentation of the sugars into alcohols or other biochemicals or biological materials in the same reaction vessel.

[0156] "Ethanogenic microorganisms" refers to microorganisms that have the ability to convert sugars or oligosaccharides into ethanol.

[0157] The term "fermented beverage" refers to any beverage produced by means of fermentation processes, such as microbial fermentation, like bacterial and / or fungal fermentation.

[0158] The term "malt" refers to any germinated grain, such as germinated barley or wheat.

[0159] The term "wine" refers to any aqueous slurry containing starch and / or sugar, such as grain flour (e.g., including crushed barley malt, crushed barley) and / or other adjuncts or combinations thereof, which is subsequently mixed with water to separate wort and waste.

[0160] The term "malt extract" refers to the unfermented liquid run-off that remains after the grain flour has been extracted during mashing preparation.

[0161] The term "approximately" refers to ±15% of the reference value.

[0162] 2. Maltopentose / maltohexasose α-amylase variants

[0163] Combinatorial variants of maltopentose / maltohexasose α-amylases are described, exhibiting high levels of performance in automated dishwashing (ADW) applications. These variants are most closely associated with an α-amylase from a Bacillus species (referred herein to as AA2560, and previously identified as BspAmy24 (SEQ ID NO:1) in WO 2018 / 184004). The mature amino acid sequence of AA2560 α-amylase is shown in SEQ ID NO:1 below:

[0164]

[0165]

[0166] The closely related maltopentosyl / maltohexasyl α-amylase comes from Bacillus species 707, referred to herein as "AA707". The mature amino acid sequence of AA707α- is shown in SEQ ID NO:2 below:

[0167]

[0168] Another closely related maltopentosyl / maltohexasyl α-amylase comes from a species of Bacillus and is called AA560. The mature amino acid sequence of AA560 is shown in SEQ ID NO:3 below:

[0169]

[0170]

[0171] Based on amino acid sequence identity, another hypothesized maltopentose / maltohexasose α-amylase originates from another Bacillus species and is referred to herein as AAI10. The mature amino acid sequence of AAI10 α-amylase is shown in SEQ ID NO:4 below:

[0172]

[0173] The comparison of these four α-amylases is shown in Figure 1 The amino acid sequence identity is summarized in Table 1. AA707, AA560, and AAI10 all share more than 80% amino acid content with AA2560.

[0174] Table 1. Amino acid sequence identity of α-amylases

[0175]

[0176] A variant of this invention is characterized by a mutation at position 91 and / or at least one mutation at the bottom of the α-amylase TIM tube structure. The residues at the bottom of the tube have a solvent-accessible surface area greater than zero and are located in or adjacent to the core β-tube structure, on the side of the tube opposite the active site, and on the side containing the N-terminus of each chain. The solvent-accessible surface area was calculated using MOE2018.01 (Chemical Computing Group, Montreal), using default parameters, and based on the AA2560 homology model constructed using MOE 2018.01 with default parameters and a 1BLI structure from pdb. The relevant residues are located at positions 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325, 362, 363, and 364, numbered with reference to SEQ ID NO:1. It can be used with Figure 2 and 3 The images in the diagram help to understand the structural significance of these residues at the bottom of the tube. In all cases, the residues are arranged at the bottom of the TIM tube structure, representing a major structural feature of α-amylase and many other enzymes. An exemplary mutation at residue 91 is the substitution of a polar residue for a charged residue, particularly a positively charged residue (such as arginine (i.e., X91R)), which in the case of AA2560 is the specific substitution of S91R.

[0177] A notable exception is that Amy707 differs from AA2560, AAI10, and AA560 (as well as Cytophaga sp. (Jeang, CL. et al. (2002) Applied and Environmental Microbiology, 68:3651-54; Genbank Accession AAA22231), Bacillus TS-23 (Lin, LL. et al. (1997) J Appl Microbiol, 82:325-34; Genbank Accession AAA63900) and other amylases) because it lacks a positively charged amino acid residue at position 28. AA2560, AAI10, and AA560, numbered according to SEQ ID NO:1, have an Arg or His side chain (position 28), while Amy707 has Asn, which cannot be positively charged (Table 2).

[0178] Table 2. Amino acid side chains at position 28 of four amylases

[0179] amylase Wild-type residues at position 28 AA707 Asn AA2560 Arg AAl10 His AA560 Arg

[0180] Therefore, in AA2560, AA560, AAI10, and many other amylases, the Ser91 mutation to Arg will result in two positively charged amino acids being positioned very close together, as shown in the image. Figure 4 The model of AA2560 shown clearly illustrates this. Without being limited to theory, it is hypothesized that the proximity of these residues can advantageously influence the activity of many amylases (such as AA2560) by repositioning the helix at positions 22-37 and 82-95 (as a result of charge-charge repulsion). In contrast, Asn28 in wild-type Amy707 may be able to form a hydrogen bond with Arg at position 91, which could lead to a tighter interaction (potentially detrimental to the activity of Amy707). SEQ ID NO:1 is used for numbering as above and below.

[0181] Exemplary mutations in the residues at the bottom of the tube include, but are not limited to, the following substitutions: X40N, X40D, X100F, X100L, X263Y, X288D, X288K, X288Q, X324R, X324N, X324M, X364L, and X364M, where “X” is an amino acid residue previously present in the wild-type parental α-amylase. Specific mutations in reference AA2560 are T40N, T40D, Y100F, Y100L, F263Y, S288D, S288K, S288Q, I324R, I324N, I324M, Y364L, and Y364M.

[0182] In different ways, the variant has one, two, three or more of the following features: N or D at position 40, F or L at position 100, Y at position 263, D, K or Q at position 288, R, N or M at position 324, or L or M at position 364.

[0183] While the combination of the mutation at position 91 and the mutation at the bottom of the cylinder produces a superior performance advantage, each mutation alone appears to produce a benefit, and some variants of the invention have a mutation at only one position / structure.

[0184] The variant may additionally have characteristic mutations in a ring comprising surface-exposed residues 167, 169, 171, 172, and 176, numbered with reference to SEQ ID NO:1. Exemplary mutations include, but are not limited to, substitutions: X167F, X169H, X171Y, X172R, X172N, and X176S, and particularly W167F, Q169H, R171Y, Q172R, Q172N, and R176S. Described differently, characteristic substitutions of the variant include F at position 167, H at position 169, Y at position 171, R or N at position 172, and / or S at position 176, numbered with reference to SEQ ID NO:1.

[0185] Variants may additionally have characteristic mutations at positions 116 and 281, which are believed to affect solubility. Exemplary mutations at these positions are substitutions for X116R and X281S, and particularly substitutions for W116R and H281S.

[0186] Variants may additionally have characteristic stable mutations at positions 190 and / or 244, numbered with reference to SEQ ID NO:1. Such mutations are well-classified and included in currently commercially available α-amylases (used in cleaning, grain processing, and textile processing). Exemplary mutations in these residues are substitutions for X190P and X244A, E, or Q, particularly E190P, S244A, S244E, and S244Q. Combinations of mutations at positions 275 and 279 with the mutation at position 190 are also of interest.

[0187] Variants may additionally have characteristic mutations at positions 1, 7, 118, 195, 202, 206, 321, 245, and 459, as numbered with reference to SEQ ID NO:1, and these variants are included in or proposed for such applications.

[0188] The variant may further include a deletion in the X1G / X2G2 motif adjacent to the calcium-binding rings corresponding to R181, G182, T183, and G184, numbered using SEQ ID NO:1. In some embodiments, the variant α-amylase comprises adjacent, paired deletions of amino acid residues corresponding to R181 and G182, or T183 and G184. The deletion corresponding to the amino acid residues of R181 and G182 may be referred to as “ΔRG”, while the deletion corresponding to the residue at position 183 (typically T, D, or H) and the amino acid residue of G184 may be appropriately referred to as “ΔTG”, “ΔDG”, “ΔHG”, etc. Both paired deletions appear to produce the same effect in the α-amylase.

[0189] Variants may further include previously described mutations for use in other α-amylases that have similar folding and / or 60% or higher amino acid sequence identity with (i) any of the well-known Bacillus α-amylases (e.g., B. lichenifomis (i.e., BLA and LAT), B. stearothermophilus (i.e., BSG), and B. amyloliquefaciens (i.e., P00692, BACAM, and BAA)) or their hybrids, (ii) any α-amylase classified as a Carbohydrate-Active Enzyme Database (CAZy) family 13 α-amylase, or (iii) any amylase previously referred to in the descriptive term “Termamyl-like”. Exemplary α-amylases include, but are not limited to, those from Bacillus species SG-1, Bacillus species 707, and α-amylases known as A7-7, SP722, DSM90 14, and KSM AP1378. Similarly, any combination of mutations described herein can produce performance advantages in these α-amylases (whether or not they are described as maltopentose / maltohexasose-producing α-amylases).

[0190] The following lists specific anticipated combination variants (numbered using SEQ ID NO:1). As mentioned above, similar variants with ΔR183-ΔT184 instead of ΔR181-ΔG182 are expected to perform as well as those described in detail.

[0191] T40-S91-Q169-ΔR181-ΔG182-T183-H281

[0192] Q172-ΔR181-ΔG182-E190-S288

[0193] Q172-ΔR181-ΔG182-S244-S288-S474

[0194] S91-Q172-ΔR181-ΔG182-E190-I324

[0195] T40-S91-ΔR181-ΔG182-E190-F263

[0196] T40-S91-ΔR181-ΔG182-S244-Y364

[0197] S91-Q172-ΔR181-ΔG182-E190-I324

[0198] S91-W116-Q172-ΔR181-ΔG182-S244-H281-S288

[0199] T40-S91-Y100-W116-Q172-ΔR181-ΔG182-S244-H281

[0200] T40-S91-Q172-ΔR181-ΔG182-S244-F263-H281

[0201] S91-Q172-ΔR181-ΔG182-E190-I324

[0202] T40-S91-Q172-ΔR181-ΔG182-E190-H281-I324

[0203] Y364-ΔR181-ΔG182

[0204] In the relevant α-amylases (including previously engineered α-amylases), mutations can be described as:

[0205] X40-X91-X169-ΔR181-ΔG182-X183-X281

[0206] X172-ΔR181-ΔG182-X190-X288

[0207] X172-ΔR181-ΔG182-X244-X288-X474

[0208] X91-X172-ΔR181-ΔG182-X190-X324

[0209] X40-X91-ΔR181-ΔG182-X190-X263

[0210] X40-X91-ΔR181-ΔG182-X244-X364

[0211] X91-X172-ΔR181-ΔG182-X190-X324

[0212] X91-X116-X172-ΔR181-ΔG182-X244-X281-X288

[0213] X40-X91-X100-X116-X172-ΔR181-ΔG182-X244-X281

[0214] X40-X91-X172-ΔR181-ΔG182-X244-X263-X281

[0215] X91-X172-ΔR181-ΔG182-X190-X324

[0216] X40-X91-X172-ΔR181-ΔG182-X190-X281-X324

[0217] X364L-ΔR181-ΔG182

[0218] Such variants include those having two, three, four, five, six or more of the following features: (a) D or N at position 40 and / or R at position 91 and (b) F at position 100, Y at position 263, D at position 288, M, N or R at position 324, and / or L at position 364, optionally combined with (c) H at position 169, M at position 183M, N or S at position 281, N or R at position 172, P at position 190, E, Q or R at position 244, R at position 474, R at position 116, optionally combined with combinations of paired omissions at positions 181 and 182 or 183 and 184.

[0219] The following lists the specific substitutions in the test variants:

[0220] T40N-S91R-Q169H-ΔR181-ΔG182-T183M-H281N

[0221] Q172R-ΔR181-ΔG182-E190P-S288D

[0222] Q172R-ΔR181-ΔG182-S244E-S288D-S474R

[0223] S91R-Q172R-ΔR181-ΔG182-E190P-I324M

[0224] T40N-S91R-ΔR181-ΔG182-E190P-F263Y

[0225] T40N-S91R-ΔR181-ΔG182-S244E-Y364L

[0226] S91R-Q172R-ΔR181-ΔG182-E190P-I324R

[0227] S91R-W116R-Q172R-ΔR181-ΔG182-S244E-H281S-S288D

[0228] T40N-S91R-Y100F-W116R-Q172N-ΔR181-ΔG182-S244Q-H281S

[0229] T40N-S91R-Q172R-ΔR181-ΔG182-S244Q-F263Y-H281S

[0230] S91R-Q172R-ΔR181-ΔG182-E190P-I324N

[0231] T40D-S91R-Q172R-ΔR181-ΔG182-E190P-H281S-I324R

[0232] Y364L-ΔR181-ΔG182

[0233] It should be understood that when α-amylase naturally possesses the mutations listed above (i.e., where wild-type α-amylase already contains residues identified as mutated), then a specific mutation does not apply to the molecule. However, other described mutations can combine with naturally occurring residues at that position.

[0234] The variant α-amylases of the present invention may also include substitutions, deletions, or additions of one or more amino acids in the amino acid sequence (e.g., 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 are expected to have similar activity to the α-amylases from which they are derived. The variant α-amylases of the present invention may also include minor deletions and / or extensions of one or more residues at their N- or C-terminus. Such minor changes are unlikely to destroy the inventive concept described herein.

[0235] The amylases of the present invention may be “precursors,” “immature,” or “full-length,” in which case they contain a signal sequence; or “mature,” in which case they lack a signal sequence. The mature form of the polypeptide is generally the most useful. Unless otherwise stated, the amino acid residue numbers used herein refer to the mature form of the corresponding amylase polypeptide.

[0236] In some embodiments, the variant α-amylase has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but less than 100%, amino acid sequence identity with SEQ ID NO:1, 2, 3, or 4.

[0237] 2.5. Nucleotides encoding variant amylase polypeptides

[0238] On the other hand, nucleic acids encoding variant α-amylase polypeptides are provided. These nucleic acids may encode a specific amylase polypeptide, or an α-amylase having a specified degree of amino acid sequence identity with a specific α-amylase.

[0239] In some embodiments, the nucleic acid encodes an α-amylase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but less than 100%, amino acid sequence identity with SEQ ID NO: 1, 2, 3, or 4. It should be understood that due to the degeneracy of the genetic code, multiple nucleic acids can encode the same polypeptide.

[0240] In some embodiments, the nucleic acid hybridizes under stringent or very stringent conditions with a nucleic acid encoding α-amylase (or a nucleic acid complementary to the nucleic acid encoding α-amylase), the α-amylase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but less than 100%, amino acid sequence identity with SEQ ID NO: 1, 2, 3, or 4.

[0241] 3. Production of variant α-amylase

[0242] The variant α-amylase of the present invention can be produced in host cells using methods well known in the art, such as by secretion or intracellular expression. Fermentation, separation, and concentration techniques are well known in the art, and conventional methods can be used to prepare concentrated solutions containing variant α-amylase peptides.

[0243] For production-scale recovery, the variant α-amylase peptide can be enriched or partially purified by removing cells using polymer flocculation, as generally described above. Alternatively, the enzyme can be enriched or purified by microfiltration and then concentrated by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme does not require enrichment or purification and can be lysed and used in whole culture medium without further processing. The enzyme can then be processed into, for example, granules.

[0244] 4. Cleaning compositions containing variant α-amylase

[0245] One aspect of the compositions and methods of the present invention relates to a cleaning composition comprising a variant of α-amylase as a component for, for example, automatic and manual dishwashing (ADW), laundry washing, and other hard surface cleaning.

[0246] 4.1. Overview

[0247] Preferably, the variant α-amylase is incorporated into detergent formulations at concentrations conventionally used for known α-amylases or lower. Since the α-amylase variant is superior in performance to any previously available variant, it is expected to provide superior performance at standard doses and similar performance at lower doses compared to existing α-amylases. Specific forms and formulations for detergent compositions containing the α-amylase of the present invention are described below.

[0248] 4.2. Automatic Dishwasher (ADW) Detergent Composition

[0249] Exemplary ADW detergent compositions include nonionic surfactants, including ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-terminated poly(oxyalkylated) alcohols, or amine oxide surfactants, present in amounts from 0% to 10% by weight; and builder agents (in the range of 5% to 60%) including phosphate builders (e.g., monophosphates, diphosphates, tripolyphosphates, other oligophosphates, sodium tripolyphosphate-STPP), and phosphate-free builders (e.g., amino acid-based compounds, including methyl-glycine-diacetic acid (MGDA) and its salts and derivatives, glutamic acid-N,N-diacetic acid (GLDA) and its salts and derivatives, iminodisuccinic acid (IDS) and its salts and derivatives). Biological, carboxymethyl inulin and its salts and derivatives, nitrotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), β-alanine diacetic acid (β-ADA) and its salts), homopolymers and copolymers of polycarboxylic acids and their partially or completely neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts, in the range of 0.5% to 50% by weight; sulfonated / carboxylated polymers in the range of about 0.1% to about 50% by weight to provide dimensional stability; drying aids in the range of about 0.1% to about 10% by weight (e.g., polyesters, especially anionic polyesters (optionally with additional monomers having 3 to 6 functional groups—typically acid, alcohol or ester functional groups—favoring polycondensation), polycarbonate-, polyurethane- And / or polyurea-polyorganosiloxane compounds or their precursors, particularly reactive cyclic carbonates and urea-type compounds; silicates (including sodium silicate or potassium silicate, such as disodium silicate, sodium metasilicate, and crystalline succinate) in the range of about 1% to about 20% by weight; inorganic bleaching agents (e.g., peroxyhydrate salts such as perborates, percarbonates, superphosphates, persulfates, and persilicates) and organic bleaching agents (e.g., organic peroxyacids, including diacid and tetraacyl peroxides, especially disperoxydodecanoic acid, disperoxytetradecanoic acid, and disperoxyhexadecanoic acid); bleaching activators (i.e., organic peracid precursors in the range of about 0.1% to about 10% by weight); bleaching catalysts (e.g., manganese triazacyclononane and... Related complexes, Co, Cu, Mn and Fe dipyridylamine and related complexes, and cobalt(III) pentamineacetate and related complexes; metal care agents (e.g., benzotriazole, metal salts and complexes, and / or silicates) in the range of about 0.1% to 5% by weight; enzymes (e.g., proteases, α-amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectic acid lyases, mannanases, keratinases, laccases, phospholipases, lysophospholipases, acyltransferases, hydrolases, aryl esterases, and mixtures thereof) in the range of about 0.01 mg to 5.0 mg per gram of the automatic dishwashing detergent composition; and enzyme stabilizer components (e.g., oligosaccharides, polysaccharides, and inorganic divalent metal salts).

[0250] Table 2 shows specific exemplary ADW compositions, of which at least some variants of the invention have been tested.

[0251] Table 2. Exemplary ADW Compositions

[0252]

[0253] 4.3. Heavy-duty liquid (HDL) laundry detergent composition

[0254] An exemplary HDL laundry detergent composition includes a detergency surfactant (10% to 40% wt / wt), comprising anionic detergent surfactants (selected from the group consisting of linear or branched or random, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and / or mixtures thereof) and optionally nonionic surfactants (selected from the group consisting of linear or branched or random, substituted or unsubstituted alkyl alkoxylated alcohols, such as C8-C18 alkyl ethoxylated alcohols and / or C6-C12 alkylphenol alkoxylates), wherein the weight ratio of the anionic detergent surfactant (having a hydrophilicity index (HIc) of 6 to 9) to the nonionic detergent surfactant is greater than 1:1. Suitable detergency surfactants also include cationic detergency surfactants (selected from the group consisting of: alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and / or mixtures thereof); zwitterionic and / or amphoteric detergency surfactants (selected from the group consisting of: alkanolamine sulfobetaine); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof.

[0255] The composition may optionally comprise a surface-enhancing polymer composed of amphiphilic alkoxylated oleo-cleaning polymers (selected from the group consisting of alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkylene imides (in the range of 0.05 wt% to 10 wt%)) and / or random graft polymers (typically comprising a hydrophilic backbone containing monomers selected from the group consisting of unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyols (such as glycerol) and mixtures thereof); and one or more hydrophobic side chains selected from the group consisting of C4-C25 alkyl groups, polypropylene, polybutene, saturated C1-C6 monocarboxylic acids of vinyl esters, C1-C6 alkyl esters of acrylic acid or methacrylic acid and mixtures thereof.

[0256] The composition may include additional polymers, such as dirt-releasing polymers (including anionic-terminated polyesters such as SRP1); polymers comprising at least one monomer unit (in a random or block configuration) selected from sugars, dicarboxylic acids, polyols, and combinations thereof; ethylene glycol terephthalate-based polymers and copolymers thereof (in a random or block configuration), such as Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, and Marloquest. SL); anti-redeposition polymers (0.1 wt% to 10 wt%, including carboxylic acid ester polymers, such as polymers containing at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesoconic acid, citraconic acid, methylene malonic acid and any mixture thereof; vinylpyrrolidone homopolymers; and / or polyethylene glycol, with a molecular weight range from 500 to 100,000 Da); cellulose polymers (including those selected from alkyl cellulose, alkylalkoxyalkyl cellulose, carboxyalkyl cellulose, alkylcarboxyalkyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose and mixtures thereof) and polymeric carboxylic acid esters (such as maleate / acrylate random copolymers or polyacrylate homopolymers).

[0257] The composition may further comprise saturated or unsaturated fatty acids, preferably saturated or unsaturated C12-C24 fatty acids (0 wt% to 10 wt%); deposition aids (examples of which include polysaccharides; preferably cellulose polymers; polypropylene dimethyl ammonium halide (DADMAC)); and copolymers of DADMAC with vinylpyrrolidone, acrylamide, imidazole, imidazoline halides and mixtures thereof (in random or block configurations); cationic guar gum; cationic cellulose, such as cationic hydroxyethyl cellulose; cationic starch; cationic polyacrylamide, and mixtures thereof.

[0258] The composition may further comprise dye transfer inhibitors, examples of which include manganese phthalocyanine, peroxidase, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone and polyvinylimidazole and / or mixtures thereof; and chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentamethylenephosphonic acid (DTPMP), hydroxyethanediphosphonic acid (HEDP), ethylenediamine N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), and diethylenetriaminepentaacetic acid (DTPA). ), propylenediaminetetraacetic acid (PDTA), 2-hydroxypyridine-N-oxide (HPNO), or methylglycine diacetic acid (MGDA), N,N-glutamic acid N,N-diacetic acid (N,N-dicarboxymethylglutamic acid tetrasodium salt (GLDA), hyponitrotriacetic acid (NTA), 4,5-dihydroxyisophenylsulfonic acid, citric acid and any salt thereof, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), and their derivatives.

[0259] The composition preferably comprises an enzyme selected from the following (typically from about 0.01 wt% to 0.03 wt% active enzyme): protease, α-amylase, lipase, cellulase, choline oxidase, peroxidase / oxidase, pectic acid lyase, mannanase, keratinase, laccase, phospholipase, lysophospholipase, acyltransferase, hydrolase, aryl esterase, and any mixture thereof. The composition may include 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 boric acid derivatives such as aromatic borate esters; or phenylboronic acid derivatives such as 4-formylphenylboronic acid).

[0260] The composition optionally includes a silicone resin or a fatty acid-based foam inhibitor; a color dye, calcium and magnesium cations, a visual signal transducer, an antifoaming agent (0.001 wt% to about 4.0 wt%) and / or a structural agent / thickener (0.01 wt% to 5 wt%, selected from the group consisting of: diglycerides and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based materials, ultrafine cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof).

[0261] The composition can be in any liquid form, such as a liquid or gel, or any combination thereof. The composition can be in any unit dosage form, such as a sachet.

[0262] 4.4. Heavy-duty dry / solid (HDD) laundry detergent compositions

[0263] Exemplary HDD laundry detergent compositions comprise detergency surfactants, including anionic detergency surfactants (e.g., linear or branched or random, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof); nonionic detergency surfactants (e.g., linear or branched or random, substituted or unsubstituted C8-C18 alkyl ethoxylates and / or C6-C12 alkylphenol alkoxylates); cationic detergency surfactants (e.g., alkyl pyridine compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphorus compounds, alkyl ternary sulfonium compounds, and mixtures thereof); zwitterionic surfactants. And / or amphoteric detergency surfactants (e.g., alkanolamine sulfobetaine), amphoteric surfactants, semi-polar nonionic surfactants; and mixtures thereof; builder agents, including phosphate-free builder agents (e.g., zeolite builder agents, examples of which include zeolite A, zeolite X, zeolite P and zeolite MAP in the range of 0 wt% to less than 10 wt%), phosphate builder agents (e.g., sodium tripolyphosphate in the range of 0 wt% to less than 10 wt%), citric acid, citrate and hypozinotriacetic acid, silicates (e.g., sodium silicate or potassium silicate or sodium metasilicate in the range of 0 wt% to less than 10 wt%), or layered silicates (SKS-6)); carbonates (e.g., in the range of 0 wt% to less than 10 wt%). Sodium carbonate and / or sodium bicarbonate in the range of t% to less than 80 wt%; and bleaching agents, including photobleaching agents (e.g., zinc phthalocyanine sulfonate, aluminum phthalocyanine sulfonate, succinate dyes and mixtures thereof); hydrophobic or hydrophilic bleaching activators (e.g., dodecyloxybenzenesulfonate, decyloxybenzenesulfonate, decyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED, nonanoyloxybenzenesulfonate-NOBS, nitrile quaternary ammonium salts and mixtures thereof); hydrogen peroxide sources (e.g., inorganic peroxide hydrate salts, examples of which include mono or tetrahydrate sodium salts of perborates, percarbonates, persulfates, superphosphates or persilicates). Pre-formed hydrophilic and / or hydrophobic peracids (e.g., percarboxylic acids and salts, percarbonic acids and salts, periodic acid and salts, peroxymonosulfate and salts, and mixtures thereof); and / or bleaching catalysts (e.g., imine bleaching promoters, examples of which include imine cations and polyions, imine zwitterions, modified amines, modified amine oxides, N-sulfonylimides, N-phosphonylimides, N-acylimides, thiadiazole dioxide, perfluoroimides, cyclic glycoketones, and mixtures thereof); and metal-containing bleaching catalysts (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations and auxiliary metal cations (e.g., zinc or aluminum) and chelates (e.g., ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and their water-soluble salts)).

[0264] The composition preferably includes enzymes such as proteases, α-amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectic acid lyases, mannanases, keratins, laccases, phospholipases, lysophospholipases, acyltransferases, hydrolases, aryl esterases, and any mixtures thereof.

[0265] The composition may optionally include additional detergent ingredients, including fragrance microencapsulations, starch-encapsulated fragrance modifiers, colorants, additional polymers (including fabric integrity and cationic polymers), dye-locking ingredients, fabric softeners, brighteners (e.g., CI fluorescent brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids, and / or cyclodextrins.

[0266] 4.5. Additional enzymes

[0267] Any of the cleaning compositions described herein may include any number of additional enzymes. Generally, the enzyme or enzyme should be compatible with the selected detergent (e.g., at optimal pH, compatibility with other enzymes and non-enzyme components, etc.), and the enzyme or enzyme should be present in an effective amount. The following enzymes are provided as examples.

[0268] Protease:

[0269] Suitable proteases include those derived from animal, plant, or microbial sources. This includes chemically modified or protein-engineered mutants, as well as naturally processed proteins. Proteases can be serine or metalloproteinases, alkaline microbial proteases, trypsin-like proteases, or chymotrypsin-like proteases. Examples of alkaline proteases are subtilisin proteases, especially those derived from Bacillus species, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (see, for example, WO 1989 / 06279). Exemplary proteases include, but are not limited to, WO 1995 / 23221, WO 1992 / 21760, WO2008 / 010925, WO 2010 / 0566356, WO 2011 / 072099, WO 2011 / 13022, WO 2011 / 140364, WO2012 / 151534, WO 2015 / 038792、WO 2015 / 089441、WO 2015 / 089447、WO 2015 / 143360、WO2016 / 001449、WO 2016 / 001450、WO 2016 / 061438、WO 2016 / 069544、WO 2016 / 069548、WO2016 / 069552、WO 2016 / 069557、WO 2016 / 069563、WO 2016 / 069569、WO 2016 / 087617、WO2016 / 087619、WO 2016 / 145428、WO 2016 / 174234、WO 2016 / 183509、WO 2016 / 202835、WO2016 / 205755、WO 2008 / 0090747、WO 2018 / 118950、WO 2018 / 169750、WO / 2018 / 118917、US5,801,039、US 5,340,735、US The metalloproteinases described in 5,500,364, US 5,855,625, RE 34606, US 5,955,340, US 5,700,676, US 6,312,936, US 6,482,628, and US 8,530,219; and the metalloproteinases described in WO 2007 / 044993, WO 2009 / 058303, WO 2009 / 058661, WO 2014 / 071410, WO 2014 / 194032, WO 2014 / 194034, WO 2014 / 194054, and WO 2014 / 194117.

[0270] Exemplary commercial proteases include, but are not limited to, MAXATASE, MAXACAL, and MAXAPEM. OXP PREFERENZ TM Proteases (e.g., P100, P110, P280, P300), EFFECTENZ TM Proteases (e.g., P1000, P1050, P2000), EXCELLENZ TM Proteases (e.g., P1000), And PURAFAST (Danisco US); ULTRA 16L ULTRA PRIMASE, DURAZYM PROGRESS and (Novozymes); BLAP TM and BLAP TM Variant (Henkel); LAVERGY TM PRO 104L (BASF), and (Bacillus subtilis protease from B. alkalophilus (Kao Corporation)). Suitable proteases include naturally occurring proteases or engineered variants that are specially selected or engineered to function at relatively low temperatures.

[0271] In specific embodiments of the compositions and methods of the present invention, the α-amylase variant is used in combination with a variant of Bacillus subtilis protease (referred to as BG46) from Bacillus giganteus, the variant having amino acid substitutions X39E, X99R, X126A, X127E, and X128G, and further having one or more additional substitutions selected from the group consisting of: N74D-M211L-N253P, R179Q-M211L-N253P, N74D-N253P, N85R-G160Q-R179Q-M211L-N212S-N253P, R179Q-N253P, G160Q-R179Q-M211L-N212S -N253P, R179Q-M211L, G160Q-R179Q-M211L-N253P, G160Q-R179Q-N212S-N 253P, N74D-M211L, M211L-N242D, G160Q-R179Q-M211L-N212S, N74D-R179Q- M211L-N253P, G160Q-R179Q-M211L, G160Q-R179Q-N253P, N74D-Q200L-M21 1L, N74D-G160Q-N212S-N253P, N74D-G160Q-M211L-N253P, G160Q-R179Q, G1 60Q-R179Q-N212S, N74D-G160Q-N253P, N74D-G160Q-R179Q-M211L-N212S- N253P, N74D-N085R-G160Q-R179Q-M211L, N74D-G160Q-M211L-N212S-N253P , N74D-N085R-N116R-Q200L-Q256E, N74D-G160Q-R179Q-N212S-N253P, N74 D-G160Q-M211L-N212S, N74D-G160Q, N74D-G160Q-R179Q-M211L-N253P, N74 D-R179Q-M211L, N74D-G160Q-N212S, N74D-G160Q-M211L, N74D-G160Q-R17 9Q-N253P, N74D, N74D-G160Q-R179Q-M211L-N212S, N74D-N085R-M211L-N21 2S, N74D-G160Q-R179Q-N212S, N74D-G160Q-R179Q-M211L, N74D-M211L-Q2 56E, N74D-G160Q-R179Q, R179Q-M211L-N212S-N253P, R179Q-M211L-N212S,N74D-N085R-R179Q-M211L-N212S, N74D-M211L-N212S, N74D-R179Q-M211L-N212S, N74D-M211L-N242D, N74D-Q200L-M211L-Q2 56E, N74D-Q200L-M211L-N242D-Q256E, N74D-Q200L, N74D-M211N-N212Q, N74D-M211N-N212Q-Q256E, N74D-M211N-Q256E, N74D- M211Q, N74D-M211Q-N212Q, N74D-M211Q-N212Q-Q256E, N74D-M211Q-Q256E, N74D-N198A-M211Q, N74D-N198A-M211Q-N212Q, N7 4D-N198A-M211Q-Q256E, N74D-N198G-M211Q, N74D-N198G-M211Q-N212Q, N74D-N198G-M211Q-Q256E, N74D-N198K-M211Q-N212Q , N74D-N198L-M211Q-N212Q, N74D-N198Q-M211Q-N212Q, N74D-N198R-M211Q-N212Q, N74D-N198T-M211Q-N212Q, N74D-N198V-M 211Q-N212Q, N74D-N212Q-Q256E, N74D-Q256E, N74D-R207Q, N74D-R207Q-M211N, N74D-R207Q-M211N-N212Q, N74D-R207Q-M211N -N212Q-Q256E, N74D-R207Q-M211N-Q256E, N74D-R207Q-M211Q, N74D-R207Q-M211Q-N212Q, N74D-R207Q-M211Q-N212Q-Q256E, N74D-R207Q-N212Q, N74D-R207Q-N212Q-Q256E, N74D-R207Q-Q256E, N74D-N198S-M211Q, and N74D-N198L-M211Q, wherein the amino acid positions are numbered according to the amino acid sequence corresponding to SEQ ID NO:5, wherein the variant has at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO:6. The amino acid sequences are shown below:

[0272] The amino acid sequence of the BG46 protease (SEQ ID NO:5):

[0273] QQTVPWGITRVQAPAVHNRGITGSGVRVAILDSGISAHSDLNIRGGASFVPGEPTTADLNGHGTHVAGTVAALNNSIGVIGVAPNAELYAVKVLGANGSGSVSGIAQGLEWAATNNMHIANMSLGSDFPSSTLERAVNYATSRDVLVIAATGNNGSGSVGYPARYANAMAVGATDQNNRRANFSQYGTGIDIVAPGVNVQSTYPGNRYVSMNGTSMATPHVAGAAALVKQRYPSWNATQIRNHLKNTATNLGNSSQFGSGLVNAEAATR

[0274] Amino acid sequence of BG46 with substitutions S39E, S99R, S126A, D127E, and F128G (SEQ ID NO:6):

[0275] QQTVPWGITRVQAPAVHNRGITGSGVRVAILDSGISAHEDLNIRGGASFVPGEPTTADLNGHGTHVAGTVAALNNSIGVIGVAPNAELYAVKVLGANGRGSVSGIAQGLEWAATNNMHIANMSLGAEGPSSTLERAVNYATSRDVLVIAATGNNGSGSVGYPARYANAMAVGATDQNNRRANFSQYGTGIDIVAPGVNVQSTYPGNRYVSMNGTSMATPHVAGAAALVKQRYPSWNATQIRNHLKNTATNLGNSSQFGSGLVNAEAATR

[0276] Lipase:

[0277] Suitable lipases include those of bacterial or fungal origin. These include chemically modified, proteolytically modified, or protein-engineered mutants. Examples of useful lipases include, but are not limited to, lipases from the genus *Humicola* (synonyms for *Thermophilic*), such as those from *H. lanuginosa* (*T. lanuginosus*) (see, for example, EP 258068 and EP 305216), and those from *H. insolens* (see, for example, WO 96 / 13580); lipases from the genus *Pseudomonas* (e.g., from *P. alcaligenes* or *P. pseudoalcaligenes*; see, for example, EP 218 272); *P. cepacia* (see, for example, EP 331 376); and *P. stutzeri* (see, for example, GB...). 1,372,034); *Pseudomonas fluorescens*; *Pseudomonas* species strain SD 705 (see, for example, WO 95 / 06720 and WO 96 / 27002); *Pseudomonas wisconsinensis* (see, for example, WO 96 / 12012); *Bacillus* lipases (e.g., from *Bacillus subtilis*; see, for example, Dartois et al. (1993), *Biochemica et Biophysica Acta*, 1131:253-360); *Bacillus stearothermophilus* (see, for example, JP 64 / 744992); or *Bacillus pumilus* (see, for example, WO 91 / 16422). Additional lipase variants to be considered for use in formulations include those described, for example, in WO 92 / 05249, WO 94 / 01541, WO95 / 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.

[0278] Exemplary commercial lipases include, but are not limited to, M1 LIPASE, LUMA FAST, and LIPOMAX (Genencor). and ULTRA (Novozymes); and LIPASE P (Amano Pharmaceutical Co., Ltd).

[0279] Polyesterase:

[0280] Suitable polyesterases may be included in the composition, such as those described in, for example, WO 01 / 34899, WO 01 / 14629, and US 6,933,140.

[0281] amylase:

[0282] The compositions of the present invention can be combined with other amylases, including other α-amylases. Such combinations are particularly desirable when different α-amylases exhibit different performance characteristics and when the combination of multiple different α-amylases results in compositions that provide the benefits of different α-amylases. Other α-amylases include commercially available α-amylases, such as, but not limited to, those listed below. and BAN TM (Novo Nordisk A / S and Novozymes); and PREFERENZ TM (From DuPont Industrial Biosciences). Exemplary α-amylases are described in WO 94 / 18314 A1, WO 2008 / 0293607, WO 2013 / 063460, WO 10 / 115028, WO 2009 / 061380 A2, WO 2014 / 099523, WO 2015 / 077126 A1, WO 2013 / 184577, WO 2014 / 164777, WO 95 / 10603, WO 95 / 26397, WO 96 / 23874, WO 96 / 23873, WO 97 / 41213, WO 99 / 19467, WO 00 / 60060, WO 00 / 29560, WO 99 / 23211、WO99 / 46399、WO 00 / 60058、WO 00 / 60059、WO 99 / 42567、WO 01 / 14532、WO 02 / 092797、WO 01 / 66712、WO 01 / 88107、WO 01 / 96537、WO 02 / 10355, WO 2006 / 002643, WO 2004 / 055178, and WO98 / 13481.

[0283] Cellulase:

[0284] Cellulases can be added to the composition. Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Suitable cellulases include those derived from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Acremonium*, for example, fungal cellulases produced from *Humicola insolens*, *Myceliophthorathermophila*, and *Fusarium oxysporum* disclosed in, for example, U.S. Patent Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO 89 / 09259. Exemplary cellulases to be used are those that have color-care benefits for textiles. Examples of such cellulases are those described in, for example, EP0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulases variants, such as those described in WO 94 / 07998; WO 98 / 12307; WO 95 / 24471; PCT / DK 98 / 00299; EP531315; and U.S. Patent Nos. 5,457,046, 5,686,593, and 5,763,254. Exemplary cellulases include those described in WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP0495257; and U.S. Provisional Applications 62 / 296,678 and 62 / 435340. Exemplary commercial cellulases include, but are not limited to, ... and PREMIUM (Novozymes); 100 200 / 220 and 2000 (Dansconis Corporation, USA); (AB Enzymes) and KAC-500(B) (Kao Corporation).

[0285] Mannanase:

[0286] Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO 2016007929; USPN 6566114, 6602842, and 6440991; and international applications PCT / US 2016 / 060850 and PCT / US 2016 / 060844.

[0287] Peroxidase / oxidase:

[0288] Suitable peroxidases / oxidases for use in the composition include those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinus* (e.g., *C. cinereus*) and their variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include, for example, GUARDZYME. TM (Novo Nordisk and Novozymes).

[0289] The detergent composition may also contain 2,6-β-D-fructan hydrolase, which is effective in removing / cleaning biofilms present on household and / or industrial textiles / clothing.

[0290] One or more detergent enzymes can be incorporated into a detergent composition by adding a single additive containing one or more enzymes, or by adding an additive containing a combination of all such enzymes. Detergent additives, whether single or in combination, can be formulated as, for example, granules, liquids, slurries, etc. Exemplary detergent additive formulations include, but are not limited to, granules (particularly dust-free granules), liquids (particularly stable liquids), or slurries.

[0291] Overhydrolytic enzymes:

[0292] The hydrolytic enzymes include those described, for example, in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO2008 / 106214, and WO 2008 / 106215.

[0293] nuclease:

[0294] Suitable nucleases include, but are not limited to, WO 2015 / 181287, WO 2015 / 155350, WO 2016 / 162556, WO 2017 / 162836, WO 2017 / 060475 (e.g., SEQ ID NO: 21), WO 2018 / 184816, WO 2018 / 177936、WO 2018 / 177938、WO 2018 / 185269、WO 2018 / 185285、WO 2018 / 177203、WO 2018 / 184817、WO 2019 / 084349、WO 2019 / 084350、WO 2019 / 081721、WO 2018 / 076800、WO Those described in 2018 / 185267, WO 2018 / 185280, and WO 2018 / 206553.

[0295] Other nucleases that can be used in combination with the variant α-amylase of this invention include those described in Nijland, R. et al. (2010) PLoS ONE 5-e15668 and Whitchurch, CB et al. (2002) Science 295:1487.

[0296] 4.6. Forms of the cleaning composition

[0297] Detergent compositions can be in any convenient form, such as strips, tablets, powders, granules, pastes, or liquids. Liquid detergents can be aqueous, typically containing up to about 70% water and 0% to about 30% organic solvents. Tight detergent gels containing about 30% or less water are also contemplated. The variant α-amylase of the present invention is compatible with known forms and formulations of detergent compositions, and specific forms and formulations are described herein.

[0298] WO 2013063460 describes numerous exemplary detergent formulations in which the α-amylase of the present invention (or in some cases, the α-amylase is identified as a component of these formulations) may be added. These include commercially available unit-dose detergent formulations / packages, such as... UltraPacks (Henkel) Quantum (Reckitt Benckiser), CLOROX TM 2Packs (Clorox), OxiClean Max Force Power Paks (Church & Dwight) Stain Release Pods ActionPacs, Platimun, And Pure essential (Procter & Gamble). Unit dosage formulations and packaging are described in, for example, US 20090209445 A1, US 20100081598 A1, US 7001878 B2, EP 1504994 B1, WO 2001085888 A2, WO 2003089562 A1, WO 2009098659 A1, WO 2009098660 A1, WO2009112992 A1, WO 2009124160 A1, WO 2009152031 A1, WO 2010059483 A1, WO2010088112 A1, WO 2010090915 A1, WO 2010135238 A1, WO 2011094687 A1, WO2011094690 A1, WO 2011127102 A1, WO 2011163428 A1, WO 2008000567 A1, WO2006045391 A1, WO 2006007911 A1, WO 2012027404 A1, EP 1740690 B1, WO 2012059336A1, US 6730646 B1, WO 2008087426 A1, WO 2010116139 A1, and WO 2012104613 A1.

[0299] 5. Carbohydrate processing using variant α-amylases

[0300] Variant α-amylases can be used in a variety of industrial carbohydrate processing applications. For example, variant α-amylases can be used in starch conversion processes, particularly in the saccharification process of starch that has undergone liquefaction. The desired end product can be any product that can be produced by the enzymatic conversion of starch substrates. For example, the desired product can be a glucose and maltose-rich syrup that can be used in other processes (e.g., the preparation of HFCS), or it can be converted into many other useful products, such as ascorbic acid intermediates (e.g., gluconate; 2-keto-L-gulonic acid; 5-keto-gluconate; 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.

[0301] Starch conversion processes can be precursors to or concurrent with fermentation processes designed to produce alcohols for fuel or drinking (i.e., drinkable alcohols). Those skilled in the art are aware of the various fermentation conditions that can be used to produce these end products. Variant α-amylases can also be used in compositions and methods of food preparation. These various uses of variant α-amylases are described in more detail below.

[0302] 5.1. Preparation of starch substrate

[0303] Methods for preparing starch substrates used in the methods disclosed herein are known. Useful starch substrates can be obtained from, for example, tubers, roots, stems, legumes, grains, or whole grains. More particularly, granular starch can be obtained from corn, cobs, wheat, barley, rye, triticale, sorghum, sago, millet, cassava, tapioca, sorghum, rice, peas, kidney beans, bananas, or potatoes. Corn starch and wheat starch are particularly considered. Starch from grains can be milled or whole and includes corn solids such as kernels, bran, and / or cobs. Starch can also be highly refined raw starch or a raw material from a starch refining process.

[0304] 5.2. Gelatinization and Liquefaction of Starch

[0305] Gelatinization typically occurs simultaneously with or after contact between the starch substrate and α-amylase, although additional liquefaction-inducing enzymes may optionally be added. In some embodiments, the starch substrate prepared as described above is slurried with water. To optimize the stability and activity of the α-amylase, depending on the characteristics of the variant α-amylase used, the pH of the slurry is typically adjusted to approximately pH 4.5–6.5, and approximately 1 mM of calcium (approximately 40 ppm of free calcium ions) may also be added. The α-amylase remaining in the slurry after liquefaction can be inactivated by a number of methods, including lowering the pH in subsequent reaction steps or removing calcium from the slurry in an enzyme-dependent manner. The starch-α-amylase slurry can be continuously pumped through a jet cooker (whose steam is heated to 105°C). The slurry is then cooled to room temperature.

[0306] 5.3. Saccharification

[0307] Liquefied starch can be saccharified into a syrup rich in low-DP (e.g., DP1+DP2) sugars using variant α-amylases, optionally in the presence of one or more other enzymes. The exact composition of the saccharified product depends on the combination of enzymes used and the type of granulated starch being processed.

[0308] Liquefaction is typically carried out as a continuous process, while saccharification is typically carried out as a batch process. Saccharification is typically most efficient at temperatures of approximately 60°C–65°C and pH of approximately 4.0–4.5 (e.g., pH 4.3), and it is necessary to cool the liquefied starch and adjust its pH. Saccharification is usually carried out in a stirred tank, which may take several hours to fill or empty. Enzymes are typically added to the dry solids at a fixed ratio (when the tank is filled) or in a single dose (at the start of the filling phase). The saccharification reaction for syrup preparation typically takes approximately 24–72 hours, e.g., 24–48 hours. For example, when the maximum or desired DE has been obtained, the reaction is stopped by heating to 85°C for 5 minutes. Further incubation will result in even lower DE as the accumulated glucose is repolymerized via enzymatic reversal reactions and / or thermodynamic equilibrium methods to isomaltose and / or other reversal products, eventually reaching approximately 90 DE.

[0309] 5.4. Isomerization

[0310] Soluble starch hydrolysates produced by treatment with a variant α-amylase can be converted into high-fructose starch-based syrups (HFSS) (such as high-fructose corn syrup (HFCS)). This conversion can be achieved using glucose isomerases, particularly glucose isomerases immobilized on a solid support. The pH is increased to approximately 6.0 to approximately 8.0, for example, pH 7.5 (depending on the isomerase), and Ca is removed by ion exchange. 2+ Suitable isomerases include IT (Novozymes); IMGI, and G993 G993 G993 liquid, and IGI. After isomerization, the mixture typically contains about 40%-45% fructose (e.g., 42% fructose).

[0311] 5.5. Fermentation

[0312] Soluble starch hydrolysates, particularly glucose-rich syrups, can be fermented by contacting the starch hydrolysates with fermenting organisms at temperatures typically around 32°C, for example, from 30°C to 35°C (for alcohol-producing yeasts). The fermentation temperature and pH will depend on the fermenting organisms. EOF products include metabolites such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, gluconate δ-lactone, sodium isoascorbate, lysine and other amino acids, ω-3 fatty acids, butanol, isoprene, 1,3-propanediol, and other biological materials.

[0313] 5.6. Combinations of variant α-amylase with other enzymes

[0314] Variant α-amylases can be combined with glucosylamylases (EC 3.2.1.3). Exemplary glucosylamylases are derived from genera such as *Trichoderma*, *Aspergillus*, *Talaromyces*, *Clostridium*, *Fusarium*, *Thielavia*, *Thermomyces*, *Athelia*, *Humicola*, *Penicillium*, *Artomyces*, *Gloeophyllum*, *Pycnoporus*, *Steccherinum*, and *Trametes*. Suitable commercial glucosylamylases include AMG 200L; AMG 300L; SAN TM SUPER and AMG TM E (Novozymes); 300 and OPTIDEX L-400 (Denisco, USA); AMIGASE TM and AMIGASE TM PLUS (DSM); G900 (EnzymeBio-Systems); and G990ZR.

[0315] Other suitable enzymes that can be used with variant α-amylases include phytases, proteases, amylopectinases, β-amylases, isoamylases, α-glucosidases, cellulases, xylanases, other hemicellulases, β-glucosidases, transferases, pectinases, lipases, keratinases, esterases, oxidoreductases, different α-amylases, or combinations thereof.

[0316] Compositions containing the α-amylase of the present invention may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., and may further contain any one or more additional enzymes listed herein, as well as buffers, salts, preservatives, water, cosolvents, surfactants, etc.

[0317] 6. Textile desizing compositions and applications

[0318] Compositions and methods for treating fabrics with amylase (e.g., desizing textiles) are also considered. Fabric treatment methods are well known in the art (see, for example, U.S. Patent No. 6,077,316). For example, the hand feel and appearance of a fabric can be improved by including contacting the fabric with an α-amylase in a solution. The fabric can be treated with the solution under pressure.

[0319] α-Amylase can be applied during or after weaving of textiles, during the desizing stage, or in one or more additional fabric processing steps. During weaving of textiles, the yarn is exposed to considerable mechanical strain. Before weaving on a loom, the warp yarns are typically coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. α-Amylase can be applied during or after weaving to remove these sizing starches or starch derivatives. After weaving, α-Amylase can be used before further fabric processing to remove the sizing coating to ensure uniform and wash-resistant results.

[0320] Alpha-amylase can be used alone or in combination with other desizing chemicals and / or desizing enzymes as a detergent additive (e.g., in aqueous compositions) to desizing fabrics, including cotton-containing fabrics. Alpha-amylase can also be used in compositions and methods for producing a stonewashed appearance on indigo-dyed denim fabrics and garments. For garment production, fabrics can be cut and sewn into clothes or garments, which are then finished. In particular, different enzymatic finishing methods have been developed for denim production. Denim garment finishing typically begins with an enzymatic desizing step, in which the garment is subjected to the action of starch-degrading enzymes to provide softness to the fabric, making the cotton more suitable for subsequent enzymatic finishing steps. Alpha-amylase can be used in methods for finishing denim garments (e.g., "biosanding process"), enzymatic desizing and providing softness to fabrics, and / or finishing processes.

[0321] 7. Compositions and methods for baking and food preparation

[0322] The compositions and methods of the present invention also relate to food compositions, including but not limited to foods, animal feeds and / or food / feed additives (containing variant α-amylase), and methods for preparing such food compositions (the method comprising mixing the variant α-amylase with one or more food ingredients), or their uses. Furthermore, the compositions and methods of the present invention relate to baking compositions (including but not limited to flour for baking, dough, baking additives and / or baking products).

[0323] 9. Brewing composition

[0324] The variant α-amylase of the present invention can be a component of a brewing composition used in a brewing process (i.e., the preparation of a fermented malt beverage). Non-fermentable carbohydrates form the majority of dissolved solids in the final beer. This residue remains because malt amylase is unable to hydrolyze the α-1,6-bonds of starch. α-amylase, optionally in combination with glucosylamylase and optionally amylopectinase and / or isoamylase, facilitates the conversion of starch into dextrins and fermentable sugars, reducing the amount of non-fermentable carbohydrates remaining in the final beer.

[0325] For all purposes, all references cited herein are incorporated herein by reference in their entirety. To further illustrate the compositions and methods and their advantages, the following specific examples are given; they should be understood as illustrative rather than limiting.

[0326] Example

[0327] Example 1. AA2560 variant

[0328] Protein expression, purification, and quantification:

[0329] A synthetic gene was prepared from the AA2560 covariant in the ΔR181 and ΔG182 (i.e., ΔRG) background and introduced into suitable Bacillus licheniformis cells using a standard procedure. All mutations were confirmed by DNA sequencing. Cells were grown for 72 hours in a medium suitable for protein expression and secretion in the Bacillus licheniformis host. The secreted protein was harvested by centrifugation. Purification was achieved by hydrophobic interaction chromatography using phenyl agarose 6 fast flow resin (GE Healthcare). The purified protein was stabilized in a standard formulation buffer (pH 8) containing HEPES (as a buffer), calcium chloride, and propylene glycol. Protein concentration was determined by simultaneous amino acid analysis, high-performance liquid chromatography (HPLC), and absorbance at 280 nm.

[0330] Enzyme performance assay:

[0331] α-Amylase activity was determined by removing stained starch stains from white melamine bricks in a detergent background. Mixed corn / rice colored starch bricks and mixed corn / rice starch bricks (catalog numbers DM277 and DM71), purchased from the Center for Testmaterials, were used to determine the cleaning activity of α-amylase. The bricks were fixed onto 96-well plates containing amylase solution diluted to the working range in aqueous buffer, and a pre-prepared detergent solution from WFKB Detergent (WFK Testgewebe, Brukergen, Germany) was added to a total volume of 300 μL. Pre-imaged melamine bricks containing stained starch stains were then fixed on top of the 96-well plates, allowing the agitator to splash the enzyme-containing detergent onto the starch stain surface. The washing reaction was carried out at 50°C with shaking at 250 rpm for 15 minutes. After the washing reaction, the melamine bricks were briefly rinsed underwater, dried, and re-imaged. α-Amylase activity is calculated as the difference in RGB (color) values ​​between the images before and after washing. The whiter the image after washing, the higher the enzyme activity. The performance index (PI) is calculated as follows:

[0332]

[0333] Performance metrics of the combined variant relative to the ΔRG variant:

[0334] Table 3 lists the cleaning performance of the variants relative to the wild-type variant in terms of performance index. DM277 is designated as Stain 1, and DM71 is designated as Stain 2.

[0335] Table 3. Properties of variants of two different stains

[0336] mutation PI-1 PI-2 ΔR181-ΔG182 -1- -1- T40N-S91R-Q169H-ΔR181-ΔG182-T183M-H281N 1.18 1.60 Q172R-ΔR181-ΔG182-E190P-S288D 1.09 1.36 Q172R-ΔR181-ΔG182-S244E-S288D-S474R 1.15 1.82 S91R-Q172R-ΔR181-ΔG182-E190P-I324M 1.26 1.95 T40N-S91R-ΔR181-ΔG182-E190P-F263Y 1.20 1.99 T40N-S91R-ΔR181-ΔG182-S244E-Y364L 1.20 2.19 S91R-Q172R-ΔR181-ΔG182-E190P-I324R 1.26 1.84 S91R-W116R-Q172R-ΔR181-ΔG182-S244E-H281S-S288D 1.29 2.19 T40N-S91R-Y100F-W116R-Q172N-ΔR181-ΔG182-S244Q-H281S 1.24 1.88 T40N-S91R-Q172R-ΔR181-ΔG182-S244Q-F263Y-H281S 1.19 1.94 S91R-Q172R-ΔR181-ΔG182-E190P-I324N 0.96 1.44 T40D-S91R-Q172R-ΔR181-ΔG182-E190P-H281S-I324R 1.04 0.99 ΔR181-ΔG182-Y364L 2.90 2.23

[0337] All variants in Table 2 performed equal to or better than those tested under one or more stain and washing conditions. Plus 12L (Novozymes).

[0338] Example 2. Additional AA2560, AA707, and AAI10 variants

[0339] To confirm the beneficial effects of the combined mutations described in Example 1 on other parental molecules, additional AA2560, AA707, and AAI10 variants were prepared and tested as described. These molecules additionally included deletions at positions 181 and 182, 183 and 184, or did not include deletions. All mutations were confirmed by DNA sequencing.

[0340] The performance of the variants was measured relative to the closest parent; that is, the combinatorial variants in the "non-deletion" molecules were measured relative to their respective wild-type molecules. The combinatorial variants in the "double-deletion" molecules were measured relative to their respective double-deletion molecules. The results are shown in… Figure 5 In the table.

[0341] The results show that, except for AA707, mutations between different parental molecules are highly transferable. The specific implementation section of this paper provides an explanation for the different behaviors of AA707 at the Ser91 position mutation.

Claims

1. A recombinant, non-naturally occurring variant α-amylase, wherein the parent amino acid sequence of said recombinant, non-naturally occurring variant α-amylase is SEQ ID NO:1, and wherein the mutation of said recombinant, non-naturally occurring variant α-amylase is selected from the group consisting of the following numbers used in SEQ ID NO:1: (a)T40N-S91R-Q169H-ΔR181-ΔG182-T183M-H281N, (b)Q172R-ΔR181-ΔG182-E190P-S288D, (c)Q172R-ΔR181-ΔG182-S244E-S288D-S474R, (d)S91R-Q172R-ΔR181-ΔG182-E190P-I324M, (e)T40N-S91R-ΔR181-ΔG182-E190P-F263Y, (f)T40N-S91R-ΔR181-ΔG182-S244E-Y364L, (g)S91R-Q172R-ΔR181-ΔG182-E190P-I324R, (h)S91R-W116R-Q172R-ΔR181-ΔG182-S244E-H281S-S288D, (i)T40N-S91R-Y100F-W116R-Q172N-ΔR181-ΔG182-S244Q-H281S, (j)T40N-S91R-Q172R-ΔR181-ΔG182-S244Q-F263Y-H281S, (k)S91R-Q172R-ΔR181-ΔG182-E190P-I324N, and (l)ΔR181-ΔG182-Y364L.

2. The recombinant, non-naturally occurring variant α-amylase as described in claim 1, wherein the mutation of the recombinant, non-naturally occurring variant α-amylase is selected from T40N-S91R-Y100F-W116R-Q172N-ΔR181-ΔG182-S244Q-H281S.

3. A detergent composition comprising a variant α-amylase as described in any one of claims 1-2.

4. A method for converting starch into oligosaccharides, the method comprising contacting starch with an effective amount of a variant α-amylase as described in any one of claims 1-2.

5. A method for removing starch stains or dirt from a surface, the method comprising contacting the surface with an effective amount of a variant α-amylase as described in any one of claims 1-2, and allowing the variant α-amylase to hydrolyze a starch component present in the starch stain to produce smaller starch-derived molecules dissolved in an aqueous composition, thereby removing the starch stain from the surface.

6. A nucleic acid encoding a variant α-amylase as described in any one of claims 1-2.

7. A host cell comprising the nucleic acid as described in claim 6.

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