Mutated α-amylase with enhanced stability in the presence of a chelating agent

Abstract

The present invention discloses mutant variant α-amylases having enhanced enzyme stability in the presence of chelating agents, methods for designing such variants, and methods of use of the resulting variants. The variant α-amylases are particularly useful for use in cleaning and desizing compositions comprising substantial amounts of chelating agents.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 745,070, filed on October 12, 2018, which is hereby incorporated by reference in its entirety. Field of the Invention

[0003] The present invention discloses mutant variant α-amylases having enhanced enzyme stability in the presence of chelating agents, methods of designing such variants, and methods of use of the resulting variants. The variant α-amylases are particularly useful for use in cleaning and desizing compositions that include substantial amounts of chelating agents. Background of the Invention

[0004] Starch consists of a mixture of amylose (15%-30% w / w) and amylopectin (70%-85% w / w). Amylose consists of a linear chain of α-1,4-linked glucose units having a molecular weight (MW) of from about 60,000 to about 800,000. Amylopectin is a branched polymer containing α-1,6 branch points every 24-30 glucose units; its MW can be as high as 100 million.

[0005] α-Amylase hydrolyzes starch, glycogen, and related polysaccharides by randomly cleaving internal α-1,4-glycosidic bonds. α-Amylases, particularly those from the genus Bacilli, have been used for a variety of different purposes, including starch liquefaction and saccharification, textile desizing, starch modification in the paper and pulp industry, brewing, baking, production of syrups for the food industry, production of feedstocks for fermentation processes, and in animal feed to increase digestibility. These enzymes can also be used to remove starch soils and stains during dishwashing and laundry washing.

[0006] Dishwashing and laundry detergent compositions, other hard surface cleaning compositions, and textile processing fluids, particularly but not limited to, typically contain substantial amounts of chelating agents, mainly to reduce hard water deposits due to the interaction of unpredictable levels of cations in local water with components present in the cleaning or desizing composition. Unfortunately, many of the most popular commercially available α-amylases rely on calcium-binding for stability and activity. Accordingly, there is a need to develop new α-amylases, as well as methods of engineering α-amylases that have high performance and high stability in the presence of chelating agents. Summary of the Invention

[0007] The compositions and methods of the present invention relate to mutant variant α-amylases having enhanced enzyme stability in the presence of chelating agents, methods of designing such variants, and methods of use of the resulting variants. Aspects and embodiments of the compositions and methods of the present invention are summarized in the following numbered paragraphs:

[0008] 1. In one aspect, a recombinant variant of a parental family 13 α - amylase is provided, wherein the variant (i) has a mutation in the side chain of an amino acid residue that is not a calcium or sodium ion ligand, (ii) wherein the mutation is capable of altering the conformational freedom, hydrogen bonding interactions, pi - stacking interactions, or van der Waals interactions of the backbone loop around the Ca 2+ -Na + -Ca 2+ site, and (iii) wherein the variant has increased stability in the presence of a predetermined amount of chelating agent as compared to the parental family 13 α - amylase lacking the mutation.

[0009] 2. In some embodiments of the variant described in paragraph 1, the mutation is at an amino acid position selected from the group consisting of:

[0010] (i) E190, V206, H210, S244, and F245, numbered using SEQ ID NO:1, or

[0011] (ii) E187, I203, H207, S241, and F242, numbered using SEQ ID NO:2.

[0012] 3. In some embodiments of the variant described in paragraph 2, the mutation is a substitution selected from the group consisting of:

[0013] (i) E190P, V206T, V206Y, H210Q, S244C, S244D, S244H, S244N, S244E, S244F, S244V, S244L, S244Q, and F245E, numbered using SEQ ID NO:1, or

[0014] (ii) E187P, I203T, I203Y, H207Q, S241C, S241D, S241H, S241N, S241E, S241F, S241V, S241L, S241Q, and F242E, numbered using SEQ ID NO:2.

[0015] 4. In some embodiments, the variant as described in any one of paragraphs 1 - 3 further comprises:

[0016] (i) a deletion or substitution at one or more residues corresponding to positions 181, 182, 183, and / or 184 in the amino acid sequence corresponding to SEQ ID NO:1;

[0017] (ii) a deletion of residues 181 and 182 or 183 and 184 corresponding to the amino acid sequence of SEQ ID NO:1;

[0018] (iii) Deletion of residues 178 and 179 or 180 and 181 corresponding to the amino acid sequence of SEQ ID NO:2;

[0019] (iv) Any single, multiple or combined mutations previously described in family 13 α -amylase; and / or

[0020] (v) N-terminal and / or C-terminal truncation.

[0021] 5. In some embodiments of the variant as described in any one of paragraphs 1-4, the variant has at least 60%, 70%, 80% or 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 and / or SEQ ID NO:2.

[0022] 6. In another aspect, there is provided a detergent composition comprising the variant amylase as described in any one of paragraphs 1-5, and the detergent composition further comprises a chelating agent.

[0023] 7. In another aspect, there is provided a composition for liquefying starch, the composition comprising the variant as described in any one of paragraphs 1-5, and the composition further comprises a chelating agent.

[0024] 8. In another aspect, there is provided a composition for desizing textiles, the composition comprising the variant as described in any one of paragraphs 1-5, and the composition further comprises a chelating agent.

[0025] 9. In another aspect, there is provided a composition for brewing or baking, the composition comprising the variant as described in any one of paragraphs 1-5, and the composition further comprises a chelating agent.

[0026] 10. In another aspect, there is provided a method for enhancing the stability of family 13 α -amylase in the presence of a chelating agent, the method comprising (i) introducing a mutation into the side chain of an amino acid residue that is not a calcium or sodium ion ligand in the parental family 13 α -amylase, (ii) wherein the mutation is capable of altering the conformational freedom, hydrogen bonding interactions, pi stacking interactions or van der Waals interactions of the backbone loop around the Ca 2+ -Na + -Ca 2+ site, and (iii) wherein the variant has increased stability in the presence of a predetermined amount of the chelating agent compared to the parental family 13 α -amylase lacking the mutation.

[0027] 11. In some embodiments of the method as described in paragraph 10, the mutation is located at the following amino acid positions, and the amino acid positions are selected from the group consisting of:

[0028] (i) E190, V206, H210, S244, and F245, numbered using SEQ ID NO:1, or

[0029] (ii) E187, I203, H207, S241, and F242, numbered using SEQ ID NO:2.

[0030] 12. In some embodiments of the method as described in paragraph 11, the mutation is a substitution selected from the group consisting of:

[0031] (i) E190P, V206T, V206Y, H210Q, S244C, S244D, S244H, S244N, S244E, S244F, S244V, S244L, S244Q, and F245E, numbered using SEQ ID NO:1, or

[0032] (ii) E187P, I203T, I203Y, H207Q, S241C, S241D, S241H, S241N, S241E, S241F, S241V, S241L, S241Q, and F242E, numbered using SEQ ID NO:2.

[0033] 13. In some embodiments of the method as described in any one of paragraphs 10-12, the variant further comprises:

[0034] (i) Deletion or substitution at one or more residues corresponding to positions 181, 182, 183, and / or 184 in the amino acid sequence of SEQ ID NO:1;

[0035] (ii) Deletion of residues 181 and 182 or 183 and 184 corresponding to the amino acid sequence of SEQ ID NO:1;

[0036] (iii) Deletion of residues 178 and 179 or 180 and 181 corresponding to the amino acid sequence of SEQ ID NO:2;

[0037] (iv) Any single, multiple, or combined mutations previously described in family 13 α-amylases; and / or

[0038] (v) N-terminal and / or C-terminal truncations.

[0039] 14. In some embodiments of the method as described in any one of paragraphs 10-13, the variant has at least 60%, 70%, 80%, or 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 and / or SEQ ID NO:2.

[0040] 15. In another aspect, there is provided a method for converting starch into oligosaccharides, the method comprising contacting the starch with an effective amount of a variant α-amylase as described in any one of paragraphs 1-5.

[0041] 16. In another aspect, there is provided 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 paragraphs 1-5 or a composition as described in paragraph 7, and allowing the polypeptide to hydrolyze the starch component present in the starch stain to produce smaller starch-derived molecules dissolved in the aqueous composition, thereby removing the starch stain from the surface.

[0042] These and other aspects and embodiments of these compositions and methods will be apparent from the present specification and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Two α-amylase models are shown, in which the α-carbon positions of the amino acid residues are highlighted with spheres, and when these positions are mutated, benefits are provided in the presence of a chelating agent. The BspAmy24 model is shown in light gray. The CspAmy2 model is shown in dark gray. Both molecules have an RG deletion. Calcium and sodium ions are shown in black.

[0044] Figure 2 The positions of the loops that surround the metal ion sites and are the source of most of the metal ligands are highlighted. The loops are shown as thicker tubes, while the rest of the structure is shown as thinner lines. Amino acids in the BspAmy24 molecule are shown in light gray. Amino acids in the CspAmy2 molecule are shown in dark gray. Both molecules have an RG deletion. Calcium and sodium ions are shown as spheres. DETAILED DESCRIPTION

[0045] Compositions and methods involving variant α-amylases having enhanced enzyme stability in the presence of a chelating agent, methods for designing such variants, and methods of using such variants are described. Such variants are particularly useful for cleaning starch stains in laundry washing, dishwashing, textile processing (e.g., desizing), and other applications in the presence of high levels of chelating agents or in special soft water environments. These and other aspects of the compositions and methods are described in detail below.

[0046] Before describing the various aspects and embodiments of the compositions and methods of the present invention, the following definitions and abbreviations are described.

[0047] 1. Definitions and Abbreviations

[0048] For the purposes of this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "a dosage" includes reference to one or more dosages and equivalents thereof known to those of ordinary skill in the art, and so forth.

[0049] This document is organized into sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for the different sections of this disclosure should not be construed as limiting.

[0050] 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 below.

[0051] 1.1. Abbreviations and Acronyms

[0052] Unless otherwise noted, the following abbreviations / acronyms have the following meanings:

[0053] DNA Deoxyribonucleic acid

[0054] EC Enzyme Commission

[0055] GA Glucoamylase

[0056] GH Total hardness

[0057] HDL High density liquid detergent

[0058] HDD Heavy duty powder detergent

[0059] HSG High suds granular detergent

[0060] HFCS High fructose corn syrup

[0061] IRS Insoluble residual starch

[0062] kDa Kilodalton

[0063] MW Molecular weight

[0064] MWU Modified Wohlgemuth unit; 1.6x10 -5 mg / MWU = Activity unit

[0065] NCBI National Center for Biotechnology Information

[0066] PI Performance index

[0067] ppm, parts per million, e.g., μg protein / g dry solids

[0068] RCF, relative centrifugal / centripetal force (i.e., x gravity)

[0069] sp., species

[0070] w / v, weight / volume

[0071] w / w, weight / weight

[0072] v / v, volume / volume

[0073] wt%, weight percentage

[0074] ℃, degree Celsius

[0075] H2O, water

[0076] dH2O or DI, deionized water

[0077] dIH2O, deionized water, Milli-Q filtered

[0078] g or gm, gram

[0079] μg, microgram

[0080] mg, milligram

[0081] kg, kilogram

[0082] μL and μl, microliter

[0083] mL and ml, milliliter

[0084] mm, millimeter

[0085] μm, micrometer

[0086] M, mole

[0087] mM, millimole

[0088] μM, micromole

[0089] U, unit

[0090] sec, second

[0091] min(s), minute

[0092] hr, hour

[0093] ETOH, ethanol

[0094] N, normal

[0095] MWCO, molecular weight cut-off

[0096] CAZy, Carbohydrate-Active enZYmes database

[0097] WT wild type

[0098] 1.2. Definitions

[0099] The term "amylase" or "amylolytic enzyme" refers to an enzyme that, among other things, is capable of catalyzing the degradation of starch. α-Amylase is a hydrolase that cleaves the α-D-(1→4) O-glycosidic bonds in starch. Generally, α-amylase (EC 3.2.1.1; α-D-(1→4)-glucan glucanohydrolase) is defined as an endo-acting enzyme that cleaves the α-D-(1→4) O-glycosidic bonds within starch molecules in a random manner, yielding polysaccharides containing three or more (1-4)-α-linked D-glucose units. In contrast, exo-acting amylolytic enzymes, such as β-amylase (EC 3.2.1.2; α-D-(1→4)-glucan maltohydrolase) and some product-specific amylases (such as maltohexaose α-amylase (EC 3.2.1.133)), cleave polysaccharide molecules from the non-reducing ends of the substrate. β-Amylase, α-glucosidase (EC 3.2.1.20; α-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; α-D-(1→4)-glucan glucohydrolase), and product-specific amylases (such as maltotetraosidase (EC 3.2.1.60) and maltohexaosidase (EC 3.2.1.98)) can produce maltooligosaccharides of specific lengths or specific maltooligosaccharides rich in syrup.

[0100] The term "starch" refers to any material composed of the complex polysaccharide carbohydrate of plants, said complex polysaccharide carbohydrate being composed of amylose and amylopectin having the formula (C6H 10 O5) x (where "X" can be any number).

[0101] With respect to polypeptides, the terms "wild type", "parent", or "reference" refer to a naturally occurring polypeptide that does not contain artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the terms "wild type", "parent", or "reference" refer to a naturally occurring polynucleotide that does not contain artificial nucleoside changes. However, note that the 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.

[0102] With respect to polypeptides, the term "variant" refers to a polypeptide that differs from a designated wild-type, parental, or reference polypeptide, in that it includes one or more naturally occurring or engineered amino acid substitutions, insertions, or deletions. Similarly, with respect to polynucleotides, the term "variant" refers to a polynucleotide that differs from a designated wild-type, parental, or reference polynucleotide in its nucleotide sequence. The characteristics of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from the context.

[0103] In the case of the α-amylase of the present invention, "activity" refers to α-amylase activity, which can be measured as described herein.

[0104] The term "performance benefit" refers to an improvement in a desired property of a molecule. Exemplary performance benefits include, but are not limited to: increased hydrolysis of starch substrates, enhanced liquefaction performance of cereal, grain, or other starch substrates, enhanced cleaning performance, enhanced thermal stability, enhanced detergent stability, enhanced storage stability, increased solubility, altered pH profile, reduced calcium dependence, enhanced stability in the presence of chelating agents, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, and increased expression. In some cases, the performance benefit is achieved at relatively low temperatures. In some cases, the performance benefit is achieved at relatively high temperatures.

[0105] The terms "chelant" and "chelating agent" are used interchangeably and refer to a compound capable of coordinating a metal ion, thereby preventing or reducing the likelihood of the metal ion interacting with other components in a solution or suspension. Exemplary chelants are described herein.

[0106] The term "metal ligand" refers to an atom of an amino acid side chain or backbone that binds to a metal and can be found, for example, in the imidazole of histidine, the thiol of cysteine, the carboxylate of aspartic acid or glutamic acid, etc.

[0107] The term "combinatorial variant" is a variant that includes two or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, deletions, and / or insertions.

[0108] The term "recombinant", when used in reference to a subject cell, nucleic acid, protein, or vector, indicates that the subject has been modified from its natural state. Thus, for example, a recombinant cell expresses a gene not found in the cell in its natural (non-recombinant) form, or expresses a native gene at a level different from that found in nature or under conditions different from those found in nature. A recombinant nucleic acid differs from the native sequence by one or more nucleotides, and / or is operably linked to a heterologous sequence, such as a heterologous promoter in an expression vector. A recombinant protein may differ from the native sequence by one or more amino acids, and / or is fused to a heterologous sequence. A vector containing a nucleic acid encoding an amylase is a recombinant vector.

[0109] The terms "recovered", "isolated", and "separated" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component that has been removed from at least one other material or component with which it is naturally associated as it exists in nature. Its "isolated" polypeptides include, but are not limited to, culture fluids containing secreted polypeptides expressed in heterologous host cells.

[0110] The term "purified" refers to a material (e.g., an isolated polypeptide or polynucleotide) in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

[0111] The term "enriched" refers to a material (e.g., an isolated polypeptide or polynucleotide) that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.

[0112] The terms "thermostable" and "thermostability" with respect to an enzyme refer to the ability of the enzyme to retain its activity after exposure to elevated temperatures. The thermostability of an enzyme (e.g., amylase) is measured by its half-life (t1 / 2) given in minutes, hours, or days, during which half of the enzyme activity is lost under defined conditions. The half-life can be calculated by measuring the residual α-amylase activity after exposure (i.e., challenged) to elevated temperatures.

[0113] The "pH range" with respect to an enzyme refers to the range of pH values under which the enzyme exhibits catalytic activity.

[0114] The terms "pH stable" and "pH stability" with respect to an enzyme relate to the ability of the enzyme to retain its activity over a wide range of pH values for a predetermined period of time (e.g., 15 min., 30 min., 1 hour).

[0115] The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein", and "peptide" and is used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as "enzymes". Amino acid sequences are represented using the conventional single-letter or three-letter codes for amino acid residues, in the standard amino-terminal-to-carboxyl-terminal orientation (i.e., N→C).

[0116] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single-stranded or double-stranded and can contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Since 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 encompass nucleotide sequences encoding specific amino acid sequences. Unless otherwise indicated, nucleic acid sequences are presented in the 5′-to-3′ orientation.

[0117] "Hybridization" refers to the process by which a nucleic acid strand forms a duplex (i.e., base pairs) with a complementary strand, as occurs during blotting hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization at 65 °C and 0.1X SSC (where 1X SSC = 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0). Hybridized double-stranded nucleic acids are characterized by a melting temperature (Tm), where half of the hybridized nucleic acid is unpaired with the complementary strand.

[0118] "Synthetic" molecules are produced by in vitro chemical or enzymatic synthesis rather than by an organism.

[0119] A "host strain" or "host cell" is an organism that has been introduced with an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., amylase). Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced from the cell.

[0120] The term "heterologous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that is not naturally present in the host cell.

[0121] The term "endogenous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that is naturally present in the host cell.

[0122] The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. The process includes both transcription and translation.

[0123] The term "specific activity" refers to the number of moles of substrate that can be converted to product per unit time by an enzyme or enzyme preparation under specific conditions. Specific activity is typically expressed as units (U) / mg protein.

[0124] As used herein, "water hardness" is a measure of the amount of minerals (such as calcium and magnesium) present in water. The United States Geological Survey classifies water as hard or soft water using the following measurement ranges (Table 1):

[0125] Table 1. Measurement ranges for classifying water by the United States Geological Survey.

[0126] Specification Hardness (mg / L) Hardness (mmol / L) Soft 0-60 0-0.60 Medium hard 61-120 0.61-1.20 Hard 121-180 1.21-1.80 Very hard >181 >1.81

[0127] A "sample" is a piece of material, such as a fabric with a stain applied thereto. The material can be, for example, a fabric made of cotton, polyester, or a mixture of natural and synthetic fibers. The sample can also be paper, such as filter paper or nitrocellulose, or a piece of rigid material, such as ceramic, metal, or glass. For α-amylase, the stain is starch-based, but can include blood, milk, ink, grass, tea, red wine, spinach, gravy, chocolate, egg, cheese, clay, pigment, oil, or a mixture of these compounds.

[0128] A "smaller sample" or "microsample" is a portion of a sample that has been cut using a single-hole punching device or a custom-made multi-hole punching device, where the pattern of the multi-hole punch matches that of a standard multi-well microtiter plate, or the portion has been otherwise removed from the sample. The sample can be a textile, paper, metal, or other suitable material. The smaller sample can have an attached stain before or after being placed in the wells of a 24-well, 48-well, or 96-well microtiter plate. The smaller sample can also be made by applying a stain to a small piece of material. For example, the smaller sample can be a contaminated fabric piece with a diameter of 5 / 8" or 0.25" or 5.5 mm. The custom-made punch is designed in such a way that it can deliver 96 samples to all the wells of a 96-well plate simultaneously. By simply loading the same 96-well plate multiple times, the device allows for the delivery of more than one sample per well. It is contemplated that the multi-hole punching device can deliver multiple samples to plates of any size, including but not limited to 24-well, 48-well, and 96-well plates. In another contemplated method, the contaminated test platform can be beads or tiles made of metal, plastic, glass, ceramic, or other suitable materials coated with a fouling substrate. Then one or more coated beads or tiles are placed in the wells of a 96-well, 48-well, or 24-well plate or a larger-sized plate, and the wells contain a suitable buffer and enzyme. In other contemplated methods, the contaminated fabric is exposed to the enzyme by spotting the enzyme solution onto the fabric, by wetting the sample attached to a holding device, or by immersing the sample in a larger solution containing the enzyme.

[0129] "Percent sequence identity" means that when aligned using the CLUSTAL W algorithm with default parameters, a particular sequence has at least a certain percentage of amino acid residues that are the same as the amino acid residues in a specified reference sequence. See Thompson et al., (1994) Nucleic Acids Res. 22:4673-4680. The default parameters of the CLUSTAL W algorithm are:

[0130]

[0131] Gaps are considered non-identical residues compared to the reference sequence.

[0132] The term "about" means ±15% of the reference value.

[0133] 2. Aspects and embodiments of the compositions and methods of the present invention

[0134] The following paragraphs detail various aspects and embodiments of the compositions and methods of the present invention.

[0135] 2.1. α-Amylase variants with increased tolerance to chelating agents

[0136] Screening was performed in two model CAZy family 13 α-amylases to identify variants with enhanced stability in the presence of 5 mM hydroxyethanediphosphonic acid (HEDP) chelating agent. Amino acid substitutions with enhanced chelate stability were found in specific structural regions of the two proteins, which are closely related to the calcium-binding site.

[0137] Without being bound by theory, it is hypothesized that the loop formed by residues 185-210 (corresponding to the amino acid sequence of BspAmy24 α-amylase (SEQ ID NO:1)) and residues 182-207 (corresponding to the amino acid sequence of CspAmy2 α-amylase (SEQ ID NO:2)) forms a 2+ -Na + -Ca 2+ feeding rack for the Ca Figure 2 ) binding site. This 185-210 loop is the source of most metal ligands, which surround the metal ion binding site and can regulate stability in the presence of chelating agents. Thus, in the presence of chelating agents, the removal of metal ions makes the 185-210 loop prone to deformation, thereby reducing the activation barrier for overall protein unfolding. Intramolecular interactions that stabilize the folded conformation of residues 185-210, and the positioning of this loop relative to spatially adjacent secondary structure regions (i.e., residues 104-184, 211-230, 236-257, and 272-284) can stabilize the folded enzyme when chelating ions are lost.

[0138] In fact, it has been found that several substitutions that alter the conformational freedom of the 185 - 210 loop or the interactions of the 185 - 210 loop within adjacent protein structural regions provide a substantial enhancement in the stability against common detergent chelating agents. It has been found that these interactions can promote the stability of two different α - amylases with less than 70% amino acid sequence identity against chelating agents, indicating that the strategy is widely applicable to CAZy family 13 α - amylases.

[0139] Specifically, the compositions and methods of the present invention comprise amino acid mutations that result in alterations in the side chains of amino acid residues that are not ligands for calcium or sodium ions, but are in the vicinity of the calcium site (i.e., within at least one atom of the atoms of the Ca 2+ -Na + -Ca 2+ metal site), and they are capable of altering the conformational freedom or hydrogen bonding, pi - stacking, or van der Waals interactions (stabilizing the folded conformation of the aforementioned structural loop around the Ca -Na 2+ -Na + -Ca 2+ site).

[0140] One model α - amylase used to illustrate the compositions and methods of the present invention is the α - amylase from a Bacillus sp. species, referred to herein as "BspAmy24 α - amylase", or simply "BspAmy24". The amino acid sequence of BspAmy24 α - amylase is shown as SEQ ID NO:1 below:

[0141]

[0142] A second model α - amylase used to illustrate the compositions and methods of the present invention is the α - amylase from a Cytophaga sp. species, referred to herein as "CspAmy2 α - amylase", or simply "CspAmy2". The amino acid sequence of CspAmy2 α - amylase is shown as SEQ ID NO:2 below:

[0143]

[0144] In some embodiments, the variant α - amylase has at least 60%, at least 70%, at least 80%, at least 85%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity with SEQ ID NO:1 and / or SEQ ID NO:2 (excluding the wild - type BspAmy24 and CspAmy2 enzymes, and their known variants).

[0145] It is known that many bacterial (and other) α - amylases share the same fold and generally benefit from the same mutations. In this case, Clustal W with default parameters can be readily used to identify the corresponding amino acid positions in other α - amylases by amino acid sequence alignment using BspAmy24 and CspAmy2. α - amylases in which the aforementioned mutations may confer a performance benefit, including any of those having a similar fold and / or having 60% or greater amino acid sequence identity to any of the well - known Bacillus amylases (such as from Bacillus lichenifomis, Bacillus stearothermophilus, Bacillus amyloliquifaciens, Bacillus species SP722, etc.), the Carbohydrate - Active Enzyme Database (CAZy) family 13 α - amylases, or any amylases hitherto referred to by the descriptive term "Termamyl - like". The reader will understand that when an α - amylase naturally has the mutations listed above (i.e., where the wild - type α - amylase already contains the residue identified as the mutation), then the specific mutation does not apply to that α - amylase. However, the other described mutations can act in combination with the naturally occurring residue at that position.

[0146] 2.2 Additional Mutations

[0147] In some embodiments, in addition to one or more of the mutations described above (e.g., in Part 2.1), the α - amylases of the present invention further comprise one or more mutations that provide further performance or stability benefits. Exemplary performance benefits include, but are not limited to: increased hydrolysis of starch substrates, enhanced liquefaction performance of cereal, grain or other starch substrates, enhanced cleaning performance, enhanced thermal stability, enhanced storage stability, increased solubility, altered pH profile, reduced calcium - dependence, increased specific activity, modified substrate specificity, modified substrate binding, modified pH - dependent activity, modified pH - dependent stability, increased oxidative stability, and increased expression. In some cases, the performance benefit is achieved at a relatively low temperature. In some cases, the performance benefit is achieved at a relatively high temperature.

[0148] In some embodiments, based on the work of Suzuki et al., (1989) J. Biol. Chem. [Journal of Biological Chemistry], 264:18933 - 938, the α - amylase variants of the present invention additionally have at least one mutation in the calcium - binding loop. Exemplary mutations include deletions or substitutions at one or more residues corresponding to positions 181, 182, 183, and / or 184 in SEQ ID NO:1 and / or 2. In certain embodiments, the mutations correspond to deletions of 181 and 182 or 183 and 184 (numbered using SEQ ID NO:1 and / or 2). Homologous residues in other α - amylases can be determined by structural alignment or by primary structure alignment.

[0149] In some embodiments, the α - amylase variants of the present invention additionally have at least one mutation known to confer performance, stability, or solubility benefits in other microbial α - amylases, including but not limited to those α - amylases having a similar fold and / or having 60% or greater amino acid sequence identity to SEQ ID NO:1 and / or 2, the Carbohydrate - Active Enzyme database (CAZy) family 13 amylases, or any amylases hitherto referred to by the descriptive term "Termamyl - like". Amino acid sequence identity can be determined using Clustal W with default parameters.

[0150] The α - amylase of the present invention can include any number of conservative amino acid substitutions. Exemplary conservative amino acid substitutions are listed in Table 2.

[0151] Table 2. Conservative Amino Acid Substitutions

[0152]

[0153]

[0154] It should be understood that some of the aforementioned conservative mutations can be generated by genetic manipulation, while others are generated by introducing synthetic amino acids into the polypeptide genetically or otherwise.

[0155] The amylases of the present invention can also be derived from any of the above - mentioned amylase variants by substituting, deleting, or adding one or several amino acids (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) in the amino acid sequence. Such variants should have the same activity as the amylase from which they are derived. Specific deletions include truncations of one or several amino acid residues, e.g., N - terminal and / or C - terminal truncations of 1, 2, 3, 4, or 5 amino acid residues.

[0156] The amylases of the present invention can be "precursor", "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 numbering used herein refers to the mature form of the corresponding amylase polypeptide. The amylase polypeptides of the present invention can also be truncated to remove N- or C-termini as long as the resulting polypeptide retains amylase activity.

[0157] The amylases of the present invention can be "chimeric", "hybrid" or "domain swap" polypeptides as they include at least a portion of a first amylase polypeptide and at least a portion of a second amylase polypeptide. The α-amylases of the present invention can further include a heterologous signal sequence, i.e., an epitope that allows for tracking or purification, etc. Exemplary heterologous signal sequences are from Bacillus licheniformis amylase (LAT), Bacillus subtilis (AmyE or AprE), and Streptomyces CelA.

[0158] 2.3. Nucleotides Encoding Variant Amylase Polypeptides

[0159] In another aspect, nucleic acids encoding variant amylase polypeptides are provided. The nucleic acids can encode a specific amylase polypeptide or an amylase having a specified degree of amino acid sequence identity to a specific amylase.

[0160] In some embodiments, the nucleic acids encode an amylase that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity to SEQ ID NO: 1 and / or 2. It should be understood that due to the degeneracy of the genetic code, multiple nucleic acids can encode the same polypeptide.

[0161] 3. Exemplary Chelating Agents

[0162] A major problem regarding the formulation and use of cleaning compounds is water hardness, mainly due to the presence of calcium, magnesium, iron, and manganese metal ions. Such metal ions interfere with the cleaning ability of surfactants and can cause substantial precipitation with surfactants. Chelating agents (chelating agents or chelants) bind to metal ions to prevent precipitation with surfactants. Unfortunately, metal ions are often essential for enzyme activity, which makes the formulation of detergent compositions an inevitable compromise.

[0163] Traditionally, the most commonly used type of chelating agent in industrial cleaning compounds has been phosphates. In the United States and Europe, phosphates are prohibited because even after sewage treatment, they re-enter the environment intact and cause oxygen depletion in waterways. Nevertheless, many countries still use phosphates, and the compositions and methods of the present invention are fully compatible with phosphate-based chelating agents.

[0164] More environmentally friendly chelating agents compatible with the compositions and methods of the present invention include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), hydroxyethane diphosphonic acid (HEDP), ethylenediamine N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamate N,N-diacetic acid (N,N-dicarboxymethylglutamate tetrasodium salt (GLDA), diethylenetriaminepentaacetic acid (DTPA), propylenediaminetetraacetic acid (PDTA), 2-hydroxypyridine-N-oxide (HPNO), nitrilotriacetic acid (NTA), 4,5-dihydroxyisophthalic acid, N-hydroxyethyl ethylenediamine triacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), citrate and gluconate (and any salts thereof) and derivatives of the above compounds.

[0165] 4. Production of variant α-amylase

[0166] 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 the variant α-amylase polypeptide.

[0167] For production-scale recovery, the variant α-amylase polypeptide can be enriched or partially purified by removing cells with 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 need to be enriched or purified, and the whole culture broth can be lysed and used without further treatment. The enzyme can then be processed into, for example, granules.

[0168] 5. Carbohydrate processing compositions and uses involving variant α-amylase

[0169] The variant α-amylase of the present invention can be used in various carbohydrate processing applications well known in the art. Such applications can involve the use of chelating agents, including but not limited to those listed, especially in cases where the local water supply is particularly difficult. Exemplary applications include fuel ethanol production, syrup production, and the production of other valuable biochemicals.

[0170] 5.1. Preparation of starch substrate

[0171] Methods for preparing starch substrates for use in the methods disclosed herein are known. Useful starch substrates can be obtained from, for example, tubers, roots, stems, legumes, grains or whole cereals. More specifically, granular starches can be obtained from maize, cobs, wheat, barley, rye, triticale, sorghum, sago, millet, cassava, tapioca, sorghum, rice, peas, beans, bananas, or potatoes. Specifically contemplated starch substrates are corn starch and wheat starch. Starches from grains can be ground or whole, and include corn solids such as kernels, bran and / or cobs. The starch can also be highly refined native starch or feedstock from a starch refining process.

[0172] 5.2. Gelatinization and liquefaction of starch

[0173] Gelatinization is typically carried out while the starch substrate is in contact with α - amylase or the starch substrate is contacted with α - amylase after gelatinization, although additional liquefaction - inducing enzymes can optionally be added. In some embodiments, the starch substrate prepared as described above is made into a slurry with water. Liquefaction can also be carried out at the liquefaction temperature or at a temperature below the liquefaction temperature, for example, in "cold cooking" or "methods without cooking".

[0174] 5.3. Saccharification

[0175] The liquefied starch can be saccharified into a syrup rich in low DP (e.g., DP1+DP2) sugars using variant α - amylase, optionally in the presence of one or more additional enzymes. The exact composition of the saccharification product depends on the combination of enzymes used and the type of granular starch being processed. Saccharification and fermentation can be carried out simultaneously or in an overlapping manner (see below).

[0176] 5.4. Isomerization

[0177] The soluble starch hydrolysis product produced by treatment with amylase can be converted into a high - fructose starch - based syrup (HFSS), such as high - fructose corn syrup (HFCS). The conversion can be achieved using glucose isomerase (especially glucose isomerase immobilized on a solid support).

[0178] 5.5. Fermentation

[0179] Soluble starch hydrolysis products (especially glucose-rich syrups) can be fermented by contacting the starch hydrolysis products with 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, glucono-delta-lactone, sodium erythorbate, lysine and other amino acids, omega-3 fatty acids, butanol, isoprene, 1,3-propanediol and other biological materials.

[0180] Ethanol-producing microorganisms include yeasts (such as Saccharomyces cerevisiae) and bacteria (such as Zymomonas moblis) that express alcohol dehydrogenase and pyruvate decarboxylase. Improved strains of ethanol-producing microorganisms are known in the art. Commercial sources of yeast include ETHANOL (LeSaffre); FERMAX TM (Martrex), Yield+ and YP3 TM (Lallemand); (Red Star); (DSM Specialties); (Alltech); and and Thrive (DuPont Industrial Biosciences). Microorganisms that produce other metabolites such as citric acid and lactic acid by fermentation are also known in the art.

[0181] 5.6. Carbohydrate processing compositions containing variant α-amylase and additional enzymes

[0182] Variant α - amylases of the present invention can be combined with glucoamylases (EC 3.2.1.3) from, for example, Trichoderma, Aspergillus, Talaromyces, Clostridium, Fusarium, Thielavia, Thermomyces, Athelia, Humicola, Penicillium, Artomyces, Gloeophyllum, Pycnoporus, Steccherinum, Trametes, etc. Commercially available glucoamylases include AMG 200L; AMG 300 L; SAN TM SUPER and AMG TM E (Novozymes); 300 and OPTIDEX L - 400 (DuPont Industrial Biosciences); AMIGASE TM and AMIGASE TM PLUS (DSM); G900 (Enzyme Bio - Systems); and G990ZR.

[0183] Other suitable enzymes that can be used with the amylase include phytase, protease, pullulanase, β - amylase, isoamylase, α - glucosidase, cellulase, xylanase, other hemicellulases, β - glucosidase, transferase, pectinase, lipase, cutinase, esterase, mannanase, oxidoreductase, different α - amylases or combinations thereof.

[0184] Compositions containing the α - amylase of the present invention can be aqueous or non - aqueous formulations, granules, powders, gels, slurries, pastes, etc., which can further contain any one or more of the additional enzymes listed herein, as well as buffers, salts, preservatives, water, co - solvents, surfactants, etc. Such compositions can act in combination with endogenous enzymes or other components already present in slurries, water baths, washing machines, food or beverage products, etc. (e.g., endogenous plant (including algae) enzymes, residual enzymes from previous processing steps, etc.).

[0185] 6. Compositions and Methods for Food and Feed Preparation

[0186] The present variant compositions and methods are also compatible with food and feed applications involving the use of chelating agents, including but not limited to those listed herein. Such applications include the preparation of food, animal feed, and / or food / feed additives. An exemplary application (primarily for the benefit of humans) is baking.

[0187] 7. Brewing compositions

[0188] The compositions and methods of the present invention are also applicable to brewing applications involving the use of chelating agents, including but not limited to those listed herein. While hard water is commonly used in the production of certain styles and varieties of beer (or its distilled products), it may be necessary to reduce the hardness of local water in order to produce other types and varieties of beer locally.

[0189] 8. Textile desizing compositions

[0190] The use of the compositions and methods of the present invention for treating fabrics (e.g., desizing fabrics) in applications involving the use of chelating agents (including but not limited to those listed herein, particularly in cases where the local water supply is particularly difficult) is also contemplated. Fabric treatment methods are well known in the art (see, for example, U.S. Patent No. 6,077,316). Fabrics can be treated with a solution under pressure.

[0191] 9. Cleaning compositions

[0192] One aspect of the compositions and methods of the present invention is cleaning compositions that contain chelating agents (including but not limited to those listed as components). Such applications include, for example, hand washing, laundry washing, dishwashing, and other hard surface cleaning. Corresponding compositions include heavy duty liquid (HDL), heavy duty dry (HDD), and hand wash (manual) laundry detergent compositions, including unit dose forms of laundry detergent compositions, and automatic dishwashing (ADW) and hand wash (manual) dishwashing compositions, including unit dose forms of dishwashing compositions.

[0193] 9.1. Overview

[0194] The amylase polypeptides of the present invention can be components of detergent compositions that contain chelating agents, either as the sole enzyme or together with other enzymes including other amylolytic enzymes. It can be included in the detergent composition in the form of dust-free granules, stabilized liquids, or protected enzymes.

[0195] The detergent composition can be in any useful form, such as powder, granule, paste, bar, or liquid. The liquid detergent can be aqueous and typically contains up to about 70% water and 0% to about 30% organic solvent. It can also be in the form of a concentrated gel type containing only about 30% water. The detergent composition comprises one or more surfactants, each of which can be anionic, non-ionic, cationic, or zwitterionic. The detergent composition can additionally comprise one or more other enzymes, such as any combination of protease, another amylolytic enzyme, mannanase, cutinase, lipase, cellulase, pectate lyase, perhydrolase, xylanase, peroxidase, and / or laccase.

[0196] Detergent compositions for specific forms containing the α-amylase of the present invention are described below. Many of these compositions can be provided in unit-dose form for ease of use. Unit-dose formulations and packages are described, for example, in US20090209445 A1, US 20100081598 A1, US 7001878 B2, EP 1504994 B1, WO 2001085888A2, WO 2003089562 A1, WO 2009098659 A1, WO 2009098660 A1, WO 2009112992 A1, WO2009124160 A1, WO 2009152031 A1, WO 2010059483 A1, WO 2010088112 A1, WO2010090915 A1, WO 2010135238 A1, WO 2011094687 A1, WO 2011094690 A1, WO2011127102 A1, WO 2011163428 A1, WO 2008000567 A1, WO 2006045391 A1, WO2006007911 A1, WO 2012027404 A1, EP 1740690 B1, WO 2012059336 A1, US 6730646 B1, WO 2008087426 A1, WO 2010116139 A1 and WO 2012104613 A1.

[0197] 9.2. Heavy-duty liquid (HDL) laundry detergent compositions

[0198] Exemplary HDL laundry detergent compositions comprise a detersive surfactant (10% to 40% wt / wt), including anionic cleaning surfactants (selected from the group consisting of linear or branched or random-chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof) and optionally nonionic surfactants (selected from the group consisting of linear or branched or random-chain, substituted or unsubstituted alkyl alkoxylated alcohols, such as C8-C18 alkyl ethoxylated alcohols and / or C6-C12 alkyl phenol alkoxylates), wherein the weight ratio of the anionic cleaning surfactant (having a hydrophilicity index (HIc) from 6.0 to 9) to the nonionic cleaning surfactant is greater than 1:1. Suitable detersive surfactants also include cationic detersive surfactants (selected from the group consisting of hydrocarbyl pyridinium compounds, hydrocarbyl quaternary ammonium compounds, hydrocarbyl quaternary phosphonium compounds, hydrocarbyl tertiary sulfonium compounds, and / or mixtures thereof); zwitterionic and / or amphoteric detersive surfactants (selected from the group of alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof.

[0199] The composition may optionally include a surface activity enhancing polymer consisting of an amphiphilic alkoxylated oil cleaning polymer, the amphiphilic alkoxylated oil cleaning polymer (selected from the group consisting of alkoxylated polymers having branched hydrophilic and hydrophobic characteristics, such as alkoxylated polyalkyleneimines (in the range of 0.05 wt% - 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, vinyl esters of saturated C1-C6 monocarboxylic acids, C1-C6 alkyl esters of acrylic or methacrylic acid, and mixtures thereof).

[0200] The composition may comprise additional polymers such as soil release polymers (including anionically terminated polyesters (e.g., SRP1); polymers comprising at least one monomer unit (in random or block configuration) selected from saccharides, dicarboxylic acids, polyols, and combinations thereof; ethylene glycol terephthalate-based polymers and their copolymers in random or block configuration, such as Repel-o-tex SF, SF-2, and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325, Marloquest SL); anti-redeposition polymers (0.1 wt% to 10 wt%, including carboxylate polymers such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof; vinylpyrrolidone homopolymers; and / or polyethylene glycols having a molecular weight range from 500 to 100,000 Da); cellulose polymers (including those cellulose polymers selected from alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, alkyl carboxyalkyl celluloses, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixtures thereof) and polymeric carboxylates (such as maleate / acrylate random copolymers or polyacrylate homopolymers).

[0201] 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; poly(diallyldimethylammonium chloride) (DADMAC)); and copolymers of DADMAC with vinylpyrrolidone, acrylamide, imidazole, imidazoline halides, and mixtures thereof (in random or block configuration); cationic guar gum; cationic cellulose, such as cationic hydroxyethyl cellulose; cationic starch; cationic polyacrylamide, and mixtures thereof.

[0202] 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, polyvinyl oxazolidone and polyvinylimidazole, and / or mixtures thereof.

[0203] The composition preferably contains enzymes (usually from about 0.01 wt% active enzyme to 0.03 wt% active enzyme) selected from the group consisting of: α-amylase (including the α-amylase of the present invention and optionally other α-amylases), protease, lipase, cellulase, choline oxidase, peroxidase / oxidase, pectate lyase, mannanase, cutinase, laccase, phospholipase, lysophospholipase, acyltransferase, perhydrolase, arylesterase, and any mixture thereof. The composition may contain enzyme stabilizers (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 phenylboric acid derivatives such as 4-formylphenylboric acid).

[0204] The composition optionally contains silicone or fatty acid-based foam inhibitors; colorant dyes, calcium and magnesium cations, visual signaling components, defoamers (from 0.001 wt% to about 4.0 wt%) and / or structuring agents / thickeners (from 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).

[0205] The composition can be in any liquid form, such as liquid or gel form, or any combination thereof. The composition can be in any unit dosage form, such as sachets.

[0206] 9.3. Heavy-duty dry / solid (HDD) laundry detergent composition

[0207] Exemplary HDD laundry detergent compositions comprise a detersive surfactant, which includes anionic detersive surfactants (e.g., linear or branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof); nonionic detersive surfactants (e.g., linear or branched or random chain, substituted or unsubstituted C8-C18 alkyl ethoxylates and / or C6-C12 alkyl phenol alkoxylates); cationic detersive surfactants (e.g., alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl triarylsulfonium compounds, and mixtures thereof); zwitterionic and / or amphoteric detersive surfactants (e.g., alkanolamine sulfobetaines), amphoteric surfactants, semi-polar nonionic surfactants; and mixtures thereof; builders, including phosphate-free builders (e.g., zeolite builders, 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 builders (e.g., sodium tripolyphosphate in the range of 0 wt% to less than 10 wt%), citric acid, citrate, and nitrilotriacetic acid, silicates (e.g., sodium silicate or potassium silicate or sodium metasilicate, or layered silicate (SKS-6) in the range of 0 wt% to less than 10 wt%); carbonates (e.g., sodium carbonate and / or sodium bicarbonate in the range of 0 wt% to less than 80 wt%); and bleaches, said bleaches including photo-bleaches (e.g., sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes, and mixtures thereof); hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine - TAED, nonanoyloxybenzenesulfonate - NOBS, nitrile quaternary ammonium salts, and mixtures thereof); hydrogen peroxide sources (e.g., inorganic peroxyhydrate salts, examples of which include the mono- or tetra-hydrate sodium salts of perborate, percarbonate, persulfate, perphosphate, or persilicate); preformed hydrophilic and / or hydrophobic peracids (e.g., percarboxylic acids and salts, percarbonic acids and salts, periodic acids and salts, peroxymonosulfuric acids and salts, and mixtures thereof); and / or bleach catalysts (e.g., imine bleach accelerators, examples of which include imine cations and polyions, imine zwitterions, modified amines, modified amine oxides, N-sulfonylimines, N-phosphonylimines, N-acylimines, thiadiazole dioxides, perfluoroimines, cyclic glyoxals, and mixtures thereof); and metal-containing bleach catalysts (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, and auxiliary metal cations, such as zinc or aluminum).

[0208] The composition preferably contains enzymes, such as proteases, amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and any mixtures thereof.

[0209] The composition may optionally contain additional detergent ingredients, including perfume microcapsules, starch-encapsulated perfume modifiers, colorants, additional polymers (including fabric integrity and cationic polymers), dye-fixing ingredients, fabric softeners, brighteners (such as C.I. fluorescent brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids, and / or cyclodextrins.

[0210] 9.4. Automatic Dishwashing (ADW) Detergent Compositions

[0211] Exemplary ADW detergent compositions contain nonionic surfactants, including ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohols or amine oxide surfactants, present in an amount from 0% to 10% by weight; builders in the range of 5% - 60%; homopolymers and copolymers of polycarboxylic acids and their partially or fully neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts, in the range from 0.5% to 50% by weight; sulfonated / carboxylated polymers in the range from about 0.1% to about 50% by weight to provide dimensional stability; drying aids in the range from about 0.1% to about 10% by weight (e.g., polyesters, especially anionic polyesters (optionally together with additional monomers having 3 to 6 functional groups conducive to polycondensation - typically acid, alcohol or ester functional groups), polycarbonate-, polyurethane- and / or polyurea-polysiloxane compounds or their precursor compounds, especially reactive cyclic carbonate and urea types); silicates in the range from about 1% to about 20% by weight (including sodium or potassium silicate, e.g., disodium silicate, sodium metasilicate and crystalline layered silicate); inorganic bleaches (e.g., peroxyhydrate salts such as perborate, percarbonate, perphosphate, persulfate and persilicate) and organic bleaches (e.g., organic peroxyacids, including diacyl and tetraacyl peroxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); bleach activators (i.e., organic peracid precursors, in the range from about 0.1% to about 10% by weight); bleach catalysts (e.g., manganese triazacyclononane and related complexes, Co, Cu, Mn and Fe bipyridylamine and related complexes, and pentaamine cobalt(III) acetate and related complexes); metal care agents in the range from about 0.1% to 5% by weight (e.g., benzotriazole, metal salts and complexes, and / or silicates); enzymes in the range from about 0.01 mg to 5.0 mg active enzyme per gram of automatic dishwashing detergent composition (e.g., protease, amylase, lipase, cellulase, choline oxidase, peroxidase / oxidase, pectate lyase, mannanase, cutinase, laccase, phospholipase, lysophospholipase, acyltransferase, perhydrolase, arylesterase, and mixtures thereof); and enzyme stabilizer components (e.g., oligosaccharides, polysaccharides, and inorganic divalent metal salts).

[0212] 9.5. Additional Enzymes

[0213] Any of the chelant-containing cleaning compositions described herein can contain any number of additional enzymes. Generally, the one or more enzymes should be compatible with the selected detergent (e.g., in terms of pH optimum, compatibility with other enzymes and non-enzyme components, etc.), and the one or more enzymes should be present in an effective amount. The following enzymes are provided as examples.

[0214] Suitable proteases include those from animal, plant or microbial sources. This includes chemically modified or protein engineered mutants, along with naturally processed proteins. The protease can be a serine protease or a metalloprotease, an alkaline microbial protease, a trypsin-like protease, or a chymotrypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from the genus Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (see, for example, WO 89 / 06279). Exemplary proteases include, but are not limited to, those described in WO 199523221, WO 199221760, WO 2008010925, WO 20100566356, WO 2011072099, WO 201113022, WO 2011140364, WO 2012151534, WO2015038792, WO 2015089441, WO 2015089447, WO 2015143360, WO 2016001449, WO2016001450, WO 2016061438, WO 2016069544, WO 2016069548, WO 2016069552, WO2016069557, WO 2016069563, WO 2016069569, WO 2016087617, WO 2016087619, WO2016145428, WO 2016174234, WO 2016183509, WO 2016202835, WO 2016205755, US20080090747, US 5,801,039, US 5,340,735, US 5,500,364, US 5,855,625, RE 34,606, US5,955,340, US 5,700,676, US 6,312,936, US 6,482,628, US 8530219, U.S. Provisional Application Nos. 62 / 331282, 62 / 343618, 62 / 351649, 62 / 437171, 62 / 437174, and 62 / 437509, and those described in PCT Application No. PCT / CN2017 / 076749; and the metalloproteases described in WO 2007 / 044993, WO 2009 / 058303, WO 2009 / 058661, WO2014 / 071410, WO 2014 / 194032, WO 2014 / 194034, WO 2014 / 194054 and WO 2014 / 194117.

[0215] Exemplary commercial proteases include, but are not limited to, MAXATASE, MAXACAL, MAXAPEM, OXP, PURAMAX TM , EXCELLASE TM , PREFERENZ TM proteases (e.g., P100, P110, P280), EFFECTENZ TM proteases (e.g., P1000, P1050, P2000), EXCELLENZ TM proteases (e.g., P1000), and PURAFAST (DuPont Industrial Biosciences); ULTRA, ULTRA, PRIMASE, DURAZYM, PROGRESS and (Novozymes); BLAP TM and BLAP TM variants (Henkel); LAVERGY TM PRO 104L (BASF), and (B. alkalophilus subtilisin protease (Kao)). Suitable proteases include naturally occurring proteases or engineered variants that are specifically selected or engineered to act at relatively low temperatures.

[0216] Suitable lipases include those of bacterial or fungal origin. This includes chemically modified, proteolytically modified or protein-engineered mutants. Examples of useful lipases include, but are not limited to, lipases from the genus Humicola (synonym Thermomyces), such as from Humicola lanuginosa (Thermomyces lanuginosus) (see, for example, EP 258068 and EP 305216), from Humicola insolens (see, for example, WO 96 / 13580); Pseudomonas lipases (e.g., from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes; see, for example, EP 218 272); Pseudomonas cepacia (see, for example, EP 331 376); Pseudomonas stutzeri (see, for example, GB1,372,034); Pseudomonas fluorescens; Pseudomonas species strain SD 705 (see, for example, WO95 / 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 contemplated for use in formulations include, for example, those described 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.

[0217] Exemplary commercial lipases include, but are not limited to, M1 LIPASE, LUMA FAST and LIPOMAX (DuPont Industrial Biosciences); and ULTRA (Novozymes A / S); and LIPASE P (Amano Pharmaceutical Co., Ltd).

[0218] Polyesterase: Suitable polyesterases can be included in the composition, such as those described in, for example, WO 01 / 34899, WO 01 / 14629, and US 6933140.

[0219] 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 the combination of multiple different α-amylases results in a composition that provides the benefits of the different α-amylases. Other α-amylases include commercially available α-amylases such as, but not limited to and BAN TM (Novo Nordisk A / S and Novozymes A / S); and PREFERENZ TM (from DuPont Industrial Biosciences). Exemplary α-amylases are described in WO 9418314A1, US 20080293607, WO 2013063460, WO 10115028, WO 2009061380 A2, WO 2014099523, WO2015077126 A1, WO 2013184577, WO 2014164777, W09510603, WO 9526397, WO 9623874, WO9623873, WO 9741213, WO 9919467, WO 0060060, WO 0029560, WO 9923211, WO 9946399, WO0060058, WO 0060059, WO 9942567, WO 0114532, WO 02092797, WO 0166712, WO 0188107, WO0196537, WO 0210355, WO 2006002643, WO 2004055178, and WO 9813481.

[0220] Suitable cellulases include those from bacterial or fungal sources. This includes chemically modified mutants or protein engineered mutants. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example, the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum as 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 contemplated for use are those that provide color care benefits to textiles. Examples of such cellulases are those described in, for example, EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998; WO 98 / 12307; WO 95 / 24471; PCT / DK 98 / 00299; EP531315; U.S. Patent Nos. 5,457,046; 5,686,593; and 5,763,254. Exemplary cellulases include those described in WO 2005054475, WO2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. Provisional Application Nos. 62 / 296,678 and 62 / 435340. Exemplary commercial cellulases include, but are not limited to PREMIUM, and (Novozymes A / S); 100, 200 / 220 and 2000 (DuPont Industrial Biosciences); and KAC-500(B) (Kao Corporation).

[0221] Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in, for example, WO2016007929; USPN 6,566,114, 6,602,842 and 6,440,991; and international application numbers PCT / US 2016 / 060850 and PCT / US 2016 / 060844. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in, for example, WO 2016007929; USPN 6566114, 6,602,842 and 6,440,991; and international application numbers PCT / US 2016 / 060850 and PCT / US 2016 / 060844.

[0222] Suitable peroxidases / oxidases contemplated for use in the composition include those of plant, bacterial or fungal origin. Also included are chemically modified mutants or protein engineered mutants. Examples of useful peroxidases include peroxidases from Coprinus (e.g., from C. cinereus) and variants thereof, 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 A / S and Novozymes A / S).

[0223] The detergent composition may also contain a 2,6-β-D-fructan hydrolase, which is effective for removing / cleaning biofilms present on household and / or industrial textiles / clothing.

[0224] One or more detergent enzymes can be included in the detergent composition by adding a separate additive containing one or more enzymes, or by adding an additive containing a combination of all these enzymes. The detergent additive, i.e., the separate additive or the combined additive, can be formulated, for example, as a granule, liquid, slurry, etc. Exemplary detergent additive formulations include, but are not limited to, granules (especially dust-free granules), liquids (especially stable liquids) or slurries.

[0225] The detergent composition can be in any convenient form, such as a bar, tablet, powder, granule, paste, or liquid. Liquid detergents can be aqueous and typically contain up to about 70% water and 0% to about 30% organic solvent. Also contemplated are concentrated detergent gels containing about 30% or less water.

[0226] Numerous exemplary detergent formulations are described in WO 2013063460, to which the α - amylase of the present invention can be added (or in some cases the α - amylase is identified as a component of these formulations). These include commercially available unit - dose detergent formulations / packaging, such as UltraPacks (Henkel), Quantum (Reckitt Benckiser), CLOROX TM 2 Packs (Clorox), OxiClean Max Force Power Paks (Church & Dwight), Stain Release, ActionPacs, and Pods (Procter & Gamble), PS.

[0227] 9.6. Methods for Evaluating Amylase Activity in Detergent Compositions

[0228] Many α - amylase cleaning assays are known in the art, including sample and microsample assays. Only a few such assays are described in the appended examples.

[0229] To further illustrate the compositions and methods and their advantages, the following specific examples are given, which should be understood to be illustrative rather than limiting.

[0230] 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, which should be understood to be illustrative rather than limiting.

[0231] Examples

[0232] Example 1: Strain and Sample Isolation

[0233] The DNA sequence encoding the protein of interest was obtained by conventional gene synthesis methods. Using standard PCR amplification techniques, a secretion signal peptide and additional 5′ and 3′ sequences were introduced for amplification and sub - cloning. Alternatively, the entire synthetic gene can be commercially produced. These DNA sequences were inserted into a bacterial vector using standard procedures for integration and secretion in Bacillus subtilis or Bacillus lichenformis cells. The construct was verified by DNA sequencing. The transformed cells were grown in a suitable expression medium for 6 - 8 hr.

[0234] The cells were separated from the protein-containing supernatant by centrifugation and then filtered through a 0.45 μm membrane (EMD Millipore). In some cases, additional purification was achieved by ion exchange chromatography using phenyl sepharose 6 fast flow resin (GE Healthcare). Protein concentration was determined by high performance liquid chromatography (HPLC) and absorbance at 280 nm.

[0235] Example 2: Stability of Variants

[0236] The relative chelator stability of the engineered variants described was evaluated by measurement based on the relative loss of activity upon incubation in a chelator solution at elevated temperature. Briefly, the enzyme was diluted in a chelator solution at a concentration of approximately 1 - 5 ppm. The chelator solution consisted of 50 mM CAPS, 0.005% Tween-80 and 5 mM hydroxyethane diphosphonic acid (HEDP), with the pH adjusted to 10.5. The enzyme-containing solution was pressurized by heating in a thermal cycler at 65 °C to 85 °C for 4 to 10 minutes. Enzyme samples in the test solution were taken both before and after pressurizing the solution at the elevated temperature. The amylase activity present in the samples was evaluated using the amylase HR assay (Megazyme). All variants included the well-known "RG deletion" (i.e., "ΔRG"), referring to residues R181 and G182 of BspAmy24 and residues R178 and G179 of CspAmy2. Table 4 shows the mutations with improvement in the two α-amylases, with their positions arranged in rows in the two molecules. Although the amino acid sequence identity of the two α-amylases is less than 70%, some mutations were found to enhance the chelator stability of both molecules.

[0237] Table 4. Mutations enhancing the chelator stability of BspAmy24 and CspAmy2 variants.

[0238]

[0239]

[0240] Example 3: Structural Analysis of Variants

[0241] Homology models of BspAmy24 and CspAmy2 α - amylases were constructed as follows. The amino acid sequence of BspAmy24 (SEQ ID NO:1) or the amino acid sequence of CspAmy2 (SEQ ID NO:2) was used as a query in MOE (Chemical Computing Group, Montreal, Canada) to retrieve the Protein Data Bank (see, e.g., Berman, H.E. et al. (2000) Nuc. Acids Res [Nucleic Acids Research]. 28:235 - 42). Bacillus licheniformis α - amylase (1BLI) was the top common hit in both retrievals. The "Homology Model" function with all default parameters was used to create models of each enzyme. X - ray diffraction crystal structures of BspAmy24 variant α - amylase and CspAmy2 variant α - amylase were also determined. These experimental structures match very well with the homology models and support the analysis performed with the homology models.

[0242] The positions of the amino acids in Table 4 are shown in Figure 1 the structural alignment of the α - amylase models. The alpha carbons of these five positions are shown as spheres for each amylase. Amino acids in the BspAmy24 α - amylase molecule (with the RG deletion described herein) are shown in light gray. Amino acids in the CspAmy2 α - amylase molecule (also with the RG deletion described herein) are shown in dark gray. Calcium and sodium ions are shown in black. As shown, the positions in Table 4 show a tight structural alignment in the two molecules.

[0243] Structural modeling also indicates that mutations at these positions may alter the interactions (stabilizing the conformation of the 185 - 210 loop and its positioning in the folded protein structure). The loop at positions 185 - 210 (BspAmy24 numbering) surrounds the Ca 2+ -Na + -Ca 2+ metal site and contains most of the ligands of these metal ions ( Figure 2 ). The amino acid mutations listed in Table 4 may alter the interactions stabilizing the 185 - 210 loop, which may be due to its location within the loop or its ability to interact with the loop as shown in Table 5.

[0244] Table 5. Localization of Amino Acid Positions in the Structure

[0245]

[0246] Further observations on the structure modeling indicate that depending on the position and conformation of the amino acids in Table 4 and the structural environment around them, the specific interaction types of the 185-210 loop can change upon mutation. The E190P / E187P mutation will stabilize the folded structure of the loop by restricting the conformational freedom of the loop to the more limited phi and psi angles available to the proline side chains. Mutations at positions 206 / 203 will alter the van der Waals and hydrophobic packing interactions with nearby regions of the protein structure. The spatial change can move the backbone such that hydrogen bonds to the adjacent strand (BspAmy24-Asn106) at that position. Tyrosine mutations can create new hydrogen bonds and / or pi-stacking with adjacent residues. The H210Q / H207Q mutation can create new hydrogen bonds with the backbone of BspAmy24-Glu212 or BspAmy24-Tyr160 or the side chain of BspAmy24-Lys185. Mutations at positions 244 / 241 can create new hydrogen bond interactions with the 185-210 loop and also alter the van der Waals interactions of Ser with BspAmy24-Lys242, which is within the viable hydrogen bond geometry at three positions on the 185-210 loop. The Phe mutation at position 245 / 242 is expected to alter the van der Waals and pi-stacking interactions with residues on the 185-210 loop (BspAmy24-Met208 / CspAmy24-Tyr205). Glu mutations can also alter the potential hydrogen bonds at the loop residues BspAmy24-Asp209, BspAmy24-Asp188, and BspAmy24-Met208. It should be noted that any of these interactions can lead to small local adjustments in the conformation of the 185-210 loop while stabilizing the overall folded structure of the loop, thereby enhancing the overall stability of the protein in the presence of the chelator.

Claims

1. A method for enhancing the stability of family 13 α -amylase in the presence of a chelating agent, said method comprising (i) introducing a mutation into a parental family 13 α -amylase in the side chain of an amino acid residue that is not a calcium or sodium ion ligand, (ii) wherein said mutation is capable of altering the conformational freedom, hydrogen bonding interactions, pi stacking interactions or van der Waals interactions of the backbone loop around the Ca 2+ -Na + -Ca 2+ site, and (iii) wherein the variant has increased stability in the presence of a predetermined amount of chelating agent compared to the parental family 13 α -amylase lacking said mutation wherein the parental family 13α-amylase is as shown in SEQ ID NO:1 or as shown in SEQ ID NO:2, wherein the mutation is a substitution selected from the group consisting of: (i) V206T, V206Y, H210Q, S244C, S244D, S244H, S244N, S244E, S244F, S244V, S244L, S244Q or F245E, numbered using SEQ ID NO:1, or (ii) E187P, I203Y, S241C, S241D, S241H, S241N, S241E, S241F, S241V, S241L, S241Q or F242E, numbered using SEQ ID NO:2, wherein the variant further comprises: (i) a deletion of residues 181 and 182 corresponding to the amino acid sequence of SEQ ID NO:1; and / or (ii) a deletion of residues 178 and 179 corresponding to the amino acid sequence of SEQ ID NO:2.

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