Alpha-amylases, baking compositions and methods

Abstract

The present invention relates to isolated polypeptides having alpha-amylase activity, polypeptides comprising an alpha-amylase catalytic domain and a carbohydrate binding module, and polynucleotides encoding the polypeptides, compositions comprising said polypeptides, as well as polynucleotides and nucleic acid constructs or vectors encoding the polypeptides, and host cells comprising the polynucleotides or constructs / vectors, as well as methods of producing and using the polypeptides.

Description

[0001] ALPHA-AMYLASES, BAKING COMPOSITIONS AND METHODS

[0002] Reference to a Sequence Listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] Field of the Invention

[0005] The present invention relates to isolated polypeptides having alpha-amylase activity, polypeptides comprising an alpha-amylase catalytic domain and a carbohydrate binding module, and polynucleotides encoding the polypeptides, compositions comprising said polypeptides, as well as polynucleotides and nucleic acid constructs or vectors encoding the polypeptides, and host cells comprising the polynucleotides or constructs / vectors, as well as methods of producing and using the polypeptides.

[0006] Description of the Related Art

[0007] W02007147835 (Novozymes A / S) discloses the preparation of dough and baked products from the dough comprising a adding a hybrid polypeptide to a dough, said hybrid comprising an alpha-amylase catalytic domain and a carbohydrate binding module, where both are identified by amino acid sequence; compositions comprising the hybrid are also disclosed.

[0008] WO2013169645 (Danisco US, Inc.) discloses several alpha-amylases and their use in saccharification or modification of starch, and the use of one alpha-amylase therein, an isolated AcAmyl or variant thereof, in a method of baking or in a baking composition.

[0009] Improved or even just alternative alpha-amylases suitable for baking are highly sought after, especially ones that have higher activity to allow a reduced dosage in use, that are capable of being produced in higheryields ortiters to improve production economy, and / orthat have other characteristics of interest.

[0010] Summary of the Invention

[0011] The present invention provides to isolated polypeptides having alpha-amylase activity, polypeptides comprising an alpha-amylase catalytic domain and a carbohydrate binding module, and polynucleotides encoding the polypeptides, compositions comprising said polypeptides, as well as polynucleotides and nucleic acid constructs or vectors encoding the polypeptides, and host cells comprising the polynucleotides or constructs / vectors, as well as methods of producing and using the polypeptides

[0012] Accordingly, in a first aspect the present invention relates to polypeptides having alphaamylase activity, wherein said polypeptide is selected from the group consisting of:

[0013] (a) a polypeptide having at least 92% sequence identity to the mature sequence of SEQ ID NO:4, at least 92% sequence identity to the mature sequence of SEQ ID NO:5, at least 92% sequence identity to the mature sequence of SEQ ID NO:6, at least 94% sequence identity to the mature sequence of SEQ ID NO:9, at least 92% sequence identity to the mature sequence of SEQ ID NO: 13, at least 75% sequence identity to the mature sequence of SEQ ID NO: 14, or at least 97% sequence identity to the mature sequence of SEQ ID NO: 15;

[0014] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1 -30 alterations, e.g. , substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or

[0015] 26 or 27 or 28 or 29 or 30 alterations;

[0016] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or

[0017] 28 or 29 or 30 alterations; and

[0018] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0019] In a second aspect, the invention relates to polypeptides comprising a catalytic domain selected from the group consisting of:

[0020] (a) a catalytic domain having at least having at least 92% sequence identity to the

[0021] GH13 alpha-amylase catalytic domain of SEQ ID NO:4, at least 92% sequence identity to the

[0022] GH13 alpha-amylase catalytic domain of SEQ ID NO:5, at least 92% sequence identity to the

[0023] GH13 alpha-amylase catalytic domain of SEQ ID NO:6, at least 94% sequence identity to the

[0024] GH13 alpha-amylase catalytic domain of SEQ ID NO:9, at least 92% sequence identity to the

[0025] GH13 alpha-amylase catalytic domain of SEQ ID NO:13, at least 75% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO: 14, or at least 97% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:15;

[0026] (b) a catalytic domain derived from any catalytic domain recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0027] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0028] (d) a fragment of any catalytic domain recited in (a), (b), or (c), wherein the fragment has alpha-amylase activity. In a third aspect, the invention relates to baking compositions comprising a baking ingredient and a polypeptide having alpha-amylase activity, wherein said polypeptide is selected from the group consisting of:

[0029] (a) a polypeptide having at least 88% sequence identity to the mature sequence of

[0030] SEQ ID NO:1 , a polypeptide having at least 87% sequence identity to the mature sequence of

[0031] SEQ ID NO:2, a polypeptide having at least 87% sequence identity to the mature sequence of

[0032] SEQ ID NO:3, a polypeptide having at least 90% sequence identity to the mature sequence of

[0033] SEQ ID NO:4, a polypeptide having at least 90% sequence identity to the mature sequence of

[0034] SEQ ID NO:5, a polypeptide having at least 90% sequence identity to the mature sequence of

[0035] SEQ ID NO:6, a polypeptide having at least 91% sequence identity to the mature sequence of

[0036] SEQ ID NO:7, a polypeptide having at least 88% sequence identity to the mature sequence of

[0037] SEQ ID NO:8, a polypeptide having at least 89% sequence identity to the mature sequence of

[0038] SEQ ID NO:9, a polypeptide having at least 91% sequence identity to the mature sequence of

[0039] SEQ ID NO: 10, a polypeptide having at least 99% sequence identity to the mature sequence of

[0040] SEQ ID NO:11 , a polypeptide having at least 99% sequence identity to the mature sequence of

[0041] SEQ ID NO: 12, a polypeptide having at least 91% sequence identity to the mature sequence of

[0042] SEQ ID NO: 13, a polypeptide having at least 75% sequence identity to the mature sequence of

[0043] SEQ ID NO: 14, a polypeptide having at least 75% sequence identity to the mature sequence of

[0044] SEQ ID NO: 15, a polypeptide having at least 75% sequence identity to the mature sequence of

[0045] SEQ ID NO: 16, a polypeptide having at least 90% sequence identity to the mature sequence of

[0046] SEQ ID NO: 17, a polypeptide having at least 90% sequence identity to the mature sequence of

[0047] SEQ ID NO:18,

[0048] (b) a polypeptide derived from the polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0049] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0050] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0051] In a fourth aspect, the invention relates to baking compositions comprising a baking ingredient and a polypeptide comprising a catalytic domain having alpha-amylase activity, wherein said catalytic domain is selected from the group consisting of:

[0052] (a) a catalytic domain having at least 88% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:1 , at least 87% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:2 at least 87% sequence identity to the GH13 alpha- amylase catalytic domain of SEQ ID NO:3, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:4, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:5, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:6, at least 91% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:7, at least 88% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:8, at least 89% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:9, at least 91% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO: 10, at least 99% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:11 , at least 99% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:12, at least 91% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:13, at least 75% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:14, at least 75% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:15, at least 75% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:16, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:17, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO: 18,

[0053] (b) a catalytic domain derived from any of the catalytic domains recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0054] (c) a catalytic domain derived from any catalytic domain recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0055] (d) a fragment of any catalytic domain recited in (a), (b), or (c), wherein the catalytic domain fragment retains its alpha-amylase activity.

[0056] A fifth aspect of the invention relates to processes for preparing a dough, or optionally a par-baked or baked product prepared from the dough, said process comprising incorporating into the dough a polypeptide as defined in any one of claims 1-6 or a baking composition as defined in any of claims 7-9 to prepare the dough, and optionally par-baking or baking the dough to produce a par-baked or baked product, respectively.

[0057] In a sixth aspect the invention relates to uses of a polypeptide having alpha-amylase activity as defined in any one of the above-mentioned aspects in a method for producing a dough, a par-baked or a baked product.

[0058] A seventh aspect of the invention relates to polynucleotides encoding a polypeptide having alpha-amylase activity as defined in any one of the earlier aspects. An eighth aspect relates to nucleic acid constructs or expression vectors comprising the polynucleotide of the seventh aspect operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0059] In a ninth aspect, the invention relates to recombinant host cells comprising the nucleic acid construct or expression vector of the seventh or eighth aspect.

[0060] A final aspect of the invention relates to methods of producing a polypeptide having alphaamylase activity, comprising cultivating the recombinant host cell of the ninth aspect under conditions conducive for production of the polypeptide and, optionally, recovering the polypeptide.

[0061] Definitions

[0062] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0063] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] Alpha-amylase catalytic domain: The term “alpha-amylase catalytic domain” is defined herein as a polypeptide having alpha-amylase catalytic activity.

[0065] Alpha-amylase catalytic activity: Endohydrolysis of 1 ,4-alpha-D-glucosidic linkages in polysaccharides containing three or more 1 ,4-alpha-linked D-glucose units. Alpha-Amylases (alpha 1 ,4-glucan 4 glucanohydrolases, EC. 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1 ,4 glucosidic oligo and polysaccharides. A number of alpha-amylases are referred to as Termamyl™ and “Termamyl™- like alpha-amylases” and are known from, e.g., WO 90 / 11352, WO 95 / 10603, WO 95 / 26397, WO 96 / 23873 and WO 96 / 23874. Another group of alpha-amylases are referred to as Fungamyl™ and “Fungamyl™-like alpha-amylases”, which are alpha-amylases related to the alpha-amylase derived from Aspergillus oryzae disclosed in WO 01 / 34784.

[0066] Carbohydrate binding module: The term “carbohydrate binding module” (CBM) is defined as a contiguous amino acid sequence within or at either the N-terminal or at the C- terminal extremity of an alpha-amylase with a discreet fold having carbohydrate-binding activity. CBMs were previously classified as cellulose-binding domains (CBDs) based on the initial discovery of several modules or domains that bound cellulose. The CAZy database provides an updated list of CBM-containing proteins, arranged by CBM family. For the purpose of this invention, Family 20 CBMs are preferred.

[0067] Catalytic domain: The term “catalytic domain” means the region of an enzyme containing the catalytic machinery of the enzyme. Increased volume of the baked product: The term increased volume of the baked product" is measured as the volume of a given loaf of bread compared to a control. The volume may be determined as known in the art.

[0068] Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native ( / .e., from the same gene) or heterologous ( / .e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

[0069] Expression: The term “expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0070] Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0071] Extension: The term “extension” means an addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the “extended” polypeptide has alphaamylase activity.

[0072] Fragment: The term “fragment” means a polypeptide, a catalytic domain, or a carbohydrate binding module having one or more amino acids absent from the amino and / or carboxyl terminus of the mature polypeptide, catalytic domain, or binding module, wherein the fragment has alpha-amylase or carbohydrate binding activity.

[0073] Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0074] Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of the present invention has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term host cell includes protoplasts created from cells.

[0075] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

[0076] Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

[0077] Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal and / or C-terminal processing (e.g., removal of signal peptide). It is well within the skilled person’s common general knowledge in the art of enzymology to determine the mature sequence of an microbially produced alpha-amylase.

[0078] Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0079] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.

[0080] Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.

[0081] Purified: The term “purified” means a nucleic acid, polypeptide or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition. In one aspect, the term purified as used herein refers to the polypeptide or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the polypeptide being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some of the soluble components of the organism and culture medium from which it is recovered. The polypeptide may be purified ( / .e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0082] Accordingly, the polypeptide may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The polypeptide may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced polypeptide. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

[0083] It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The polypeptide of the present invention is preferably in a substantially pure form i.e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

[0084] Recover: The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and / or purification of the polypeptide.

[0085] Sequence difference: The term "sequence difference" means the percent of amino acid differences between a polypeptide and the polypeptide of SEQ ID NO: 3, and is calculated as follows:

[0086] (Different Residues x 100) / (Length of SEQ ID NO: 3) wherein the different residues comprise any substitution, deletion, or insertion (e.g., an extension at the N-terminus and / or C-terminus) in the sequence.

[0087] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0088] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0089] Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0090] Subsequence: The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and / or 3' end of a mature polypeptide coding sequence, wherein the subsequence encodes a fragment having alpha-amylase activity.

[0091] Variant: The term “variant” means a polypeptide having alpha-amylase or carbohydrate binding activity comprising a man-made mutation, i.e., a substitution, insertion (including extension), and / or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0092] Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, ammo acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

[0093] Detailed Description of the Invention

[0094] Polypeptides Having Alpha-amylase Activity

[0095] In a first aspect the invention relates to polypeptides having alpha-amylase activity, wherein said polypeptide is selected from the group consisting of:

[0096] (a) a polypeptide having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:4, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:5, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:6, at least 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:9, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 13, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 14, or at least 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:15;

[0097] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1 -30 alterations, e.g. , substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0098] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0099] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0100] In a second aspect, the invention relates to polypeptides having alpha-amylase activity, which are:

[0101] (a) a polypeptide having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:4; or (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1 -30 alterations, e.g. , substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0102] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0103] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity

[0104] Preferably, the polypeptide of the first aspect is:

[0105] (a) a polypeptide having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:5; or

[0106] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0107] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0108] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0109] Preferably, the polypeptide of the first aspect is:

[0110] (a) a polypeptide having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:6; or

[0111] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0112] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0113] Preferably, the polypeptide of the first aspect is:

[0114] (a) a polypeptide having at least 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:9; or

[0115] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0116] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0117] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0118] Preferably, the polypeptide of the first aspect is:

[0119] (a) a polypeptide having at least 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 13; or

[0120] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0121] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0122] Preferably, the polypeptide of the first aspect is:

[0123] (a) a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:14; or

[0124] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0125] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0126] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0127] Preferably, the polypeptide of the first aspect is:

[0128] (a) a polypeptide having at least 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 15; or

[0129] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0130] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0131] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0132] Preferably, the polypeptide of the first aspect is comprising, consisting essentially of, or consisting of the mature part of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15. A second aspect of the invention relates to polypeptides comprising a catalytic domain selected from the group consisting of:

[0133] (a) a catalytic domain having at least having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:4, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:5, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:6, at least 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:9, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:13, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO: 14, or at least 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:15;

[0134] (b) a catalytic domain derived from any catalytic domain recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0135] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0136] (d) a fragment of any catalytic domain recited in (a), (b), or (c), wherein the fragment has alpha-amylase activity.

[0137] Preferably, the polypeptide of the second aspect is further comprising a native or heterologous carbohydrate binding module, preferably a native or heterologous carbohydrate binding module of CaZy Family 20.

[0138] Preferably, the polypeptide of the first or second aspect is isolated or purified.

[0139] In another aspect, the polypeptide is derived by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from a mature polypeptide by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived by substitution, deletion or addition of one or several amino acids. In some embodiments, the polypeptide is a variant comprising a substitution, deletion, and / or insertion at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 3 is up to 15, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an ammo-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding module.

[0140] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for alphaamylase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver ef al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and / or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides / proteins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and / or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

[0141] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0142] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness etal., 1999, Nature Biotechnology 'll-. 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide. Sources of Polypeptides Having Alpha-amylase Activity

[0143] A polypeptide having alpha-amylase activity of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide of the invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0144] It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0145] The polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0146] The dough

[0147] As used herein “dough” means any dough used to prepare a baked product, in particular a bread.

[0148] According to the present invention, the dough used to prepare a baked product may be made from any suitable dough ingredients comprising flour.

[0149] The flour may be from any baking grain known in the art, such as, wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum flour, potato flour, soy flour, and any combinations thereof (e.g., wheat flour combined with one of the other flour sources; or rice flour combined with one of the other flour sources).

[0150] In a preferred embodiment, the flour is wheat flour.

[0151] In a preferred embodiment, at least 10% (w / w) or more of the total flour content is wheat flour, e.g., at least 15 % or more of the total flour content is wheat flour, e.g., at least 20% or more of the total flour content is wheat flour, e.g., at least 25% or more of the total flour content is wheat flour, e.g., at least 30% or more of the total flour content is wheat flour, e.g., at least 35 % or more of the total flour content is wheat flour, e.g., at least 40% or more of the total flour content is wheat flour, e.g., at least 45% or more of the total flour content is wheat flour, e.g., at least 50% or more of the total flour content is wheat flour, e.g., at least 55% or more of the total flour content is wheat flour, e.g., at least 60% or more of the total flour content is wheat flour, e.g., at least 65% or more of the total flour content is wheat flour, e.g., at least 70% or more of the total flour content is wheat flour, e.g., at least 75% or more of the total flour content is wheat flour, e.g., at least 80% or more of the total flour content is wheat flour, e.g., at least 85% or more of the total flour content is wheat flour, e.g., at least 90% or more of the total flour content is wheat flour, e.g., at least 95% or more of the total flour content is wheat flour, e.g., 100% of total the flour is wheat flour.

[0152] The dough of the invention is normally a leavened dough or a dough to be subjected to leavening. The dough may be leavened in various ways, such as by adding dough ingredients such as chemical leavening agents, e.g., sodium bicarbonate or by adding a leaven (fermenting dough), but it is preferred to leaven the dough by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), e.g., a commercially available strain of S. cerevisiae.

[0153] The dough of the invention may typically comprise some added sugar as the method according to the invention is able to reduce the amount of added sugar, but normally a partially reduction of sugar is obtained.

[0154] The dough may also comprise other conventional dough ingredients, e.g., proteins, such as milk powder, gluten, and soy; eggs (either whole eggs, egg yolks or egg whites); an oxidant such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; an amino acid such as L-cysteine; a salt such as sodium chloride, calcium acetate, sodium sulphate, calcium sulphate, diluents such as silica dioxide, and starch of different origins. Still other conventional ingredients include hydrocolloids such as CMC, guar gum, xanthan gum, locust bean gum, etc.

[0155] The dough ingredients may typically comprise fat (triglyceride) and / or oil and / or shortenings, in particular oil such as sunflower oil or rapeseed oil.

[0156] The dough may be prepared applying any conventional mixing process, such as the continuous mix process, straight-dough process, or the sponge and dough method.

[0157] The present invention is particularly useful for preparing dough and baked products in industrialized processes in which the dough used to prepare the baked products are prepared mechanically using automated or semi-automated equipment.

[0158] The process of preparing bread generally involves the sequential steps of dough making, sheeting or dividing, shaping or rolling, and proofing the dough, which steps are well known in the art.

[0159] As used herein, “baked product” means any kind of baked product including bread types such as pan bread, toast bread, open bread, pan bread with and without lid, buns, Fino bread, Hammam bread, Samoli bread, baguettes, brioche hamburger buns, rolls, brown bread, whole meal bread, rich bread, bran bread, flat bread, tortilla, biscuits, and any variety thereof. According to the present invention, the baked product may also be a cake or any patisserie product as known in the art. Baking compositions

[0160] One aspect of the invention relates to baking compositions comprising a baking ingredient and a polypeptide having alpha-amylase activity, wherein said polypeptide is selected from the group consisting of:

[0161] (a) a polypeptide having at least 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:1 , a polypeptide having at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:2, a polypeptide having at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:3, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:4, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:5, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:6, a polypeptide having at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:7, a polypeptide having at least 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:8, a polypeptide having at least 89%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:9, a polypeptide having at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 10, a polypeptide having at least 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:11 , a polypeptide having at least 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 12, a polypeptide having at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:13, a polypeptide having at least 75%, 80%, 85%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:14, a polypeptide having at least 75%, 80%, 85%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 15, a polypeptide having at least 75%, 80%, 85%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:16, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 17, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:18, (b) a polypeptide derived from the polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0162] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0163] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0164] Another aspect of the invention relates to baking compositions comprising a baking ingredient and a polypeptide comprising a catalytic domain having alpha-amylase activity, wherein said catalytic domain is selected from the group consisting of:

[0165] (a) a catalytic domain having at least 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:1 , at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:2 at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:3, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:4, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:5, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:6, at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:7, at least 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:8, at least 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:9, at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO: 10, at least 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:11 , at least 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:12, at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:13, at least 75%, 80%, 85% 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:14, at least 75%, 80%, 85% 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:15, at least 75%, 80%, 85% 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:16, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:17, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:18,

[0166] (b) a catalytic domain derived from any of the catalytic domains recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0167] (c) a catalytic domain derived from any catalytic domain recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0168] (d) a fragment of any catalytic domain recited in (a), (b), or (c), wherein the catalytic domain fragment retains its alpha-amylase activity.

[0169] The polypeptides having alpha-amylase of the invention as well as any additional enzyme(s) may be added to flour or dough in any suitable form, such as, e.g., in the form of a liquid, in particular a stabilized liquid, or it may be added to flour or dough as a substantially dry powder or granulate. Preferably, the polypeptide comprising a catalytic domain having alphaamylase activity further comprises a native or heterologous carbohydrate binding module, preferably a native or heterologous carbohydrate binding module of CaZy Family 20.

[0170] Preferred baking ingredients in the baking compositions comprise baking flour, fat and / or a leavening agent.

[0171] Preferably, the baking compositions further comprise an additional enzyme, more preferably the additional enzyme is selected from maltogenic alpha-amylase, raw starch degrading alpha-amylase, beta-amylase, xylanase, lipase, phospholipase, glucoamylase, protease, cellulase,

[0172] Baking composition granulates may be produced, e.g., as disclosed in US Patent No. 4,106,991 and US Patent No. 4,661 ,452. Liquid enzyme preparations may, for instance, be stabilized by adding a sugar or sugar alcohol or lactic acid according to established procedures. Other enzyme stabilizers are well-known in the art.

[0173] The enzyme(s) may be added to the bread dough ingredients in any suitable manner, such as individual components (separate or sequential addition of the enzymes) or addition of the enzymes together in one step or one composition.

[0174] The baking composition may contain other dough-improving and / or bread-improving additives, e.g., any of the additives, including enzymes, mentioned above. The baking composition may be, e.g., a dough composition, a flour composition, a flour pre-mix, or a bread improver.

[0175] Preferably, the baking compositions of the invention also comprise one or more additional enzyme selected from the group consisting of a alpha-amylase, maltogenic amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellulytic enzyme, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1 ,4-alpha- maltotetrahydrolase, glucanase, galactanase, alpha-galactosidase, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase.

[0176] Preferably, the baking compositions of the invention also comprise flour, sugar, yeast, salt and / or fat.

[0177] It will often be advantageous to provide the enzymes used in the treatment of the present invention in admixture with other ingredients used to improve the properties of baked products. These baking compositions are commonly known in the art as "pre-mixes," which usually comprise flour.

[0178] Hence, in a further aspect, the present invention relates to a bread premix for improving the quality of dough by reducing the amount of added sugar, which premix comprises the enzymes of the present invention.

[0179] In one embodiment, the present invention further relates to a bread pre-mix comprising the enzymes of the present invention and flour, such as, flour from grains, such as, wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, and combinations thereof.

[0180] In another embodiment, the present invention relates to a bread pre-mix comprising the enzymes of the present invention and flour, such as, flour from grains, such as, wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum, soy flour, and combinations thereof, and one or more additional enzymes, as previously described.

[0181] The pre-mix may be in the form of a granulate or agglomerated powder, e.g., wherein typically 95 % (by weight) of the granulate or agglomerated powder has a particle size between 25 and 500 pm.

[0182] Granulates and agglomerated powders may be prepared by conventional methods, e.g., by spraying the enzymes onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g. a salt (such as NaCI or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy. Additional enzymes

[0183] Optionally, one or more additional enzymes may be comprised in the baking compositions or baking processes or methods of the invention, such as one or more additional alpha-amylase, maltogenic amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellulytic enzyme, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1 ,4-alpha-maltotetrahydrolase, glucanase, galactanase, alpha-galactosidase, betagalactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase may be used together with the enzyme composition according to the invention.

[0184] The additional enzyme(s) may be of any origin, including mammalian, plant, and microbial (bacterial, yeast or fungal) origin.

[0185] The maltogenic alpha-amylase (EC 3.2.1.133) may be from Bacillus. A maltogenic alpha-amylase from B. stearothermophilus strain NCIB 11837 is commercially available from Novozymes A / S under the tradename Novamyl®.

[0186] The maltogenic alpha-amylase may also be a variant of the maltogenic alpha-amylase from B. stearothermophilus as disclosed in, e.g., WO1999 / 043794; W02006 / 032281 ; or W02008 / 148845, e.g., Novamyl® 3D.

[0187] An anti-staling amylase for use in the invention may also be an amylase (glucan 1 ,4- alpha-maltotetrahydrolase (EC 3.2.1 .60)) from Pseudomonas saccharophilia or variants thereof, such as any of the amylases disclosed in W01999 / 050399, W02004 / 111217 or W02005 / 003339.

[0188] The glucose oxidase may be a fungal glucose oxidase, in particular an Aspergillus niger glucose oxidase (such as GLUZYME®, available from Novozymes A / S).

[0189] The xylanase which may be of microbial origin, e.g., derived from a bacterium or fungus, such as a strain of Aspergillus, in particular of A. aculeatus, A. niger, A. awamori, or A. tubigensis, from a strain of Trichoderma, e.g. T. reesei, or from a strain of Humicola, e.g., H. insolens.

[0190] Suitable commercially available xylanase preparations for use in the present invention include PANZEA BG, PENTOPAN MONO BG and PENTOPAN 500 BG (available from Novozymes A / S), GRINDAMYL POWERBAKE (available from Danisco), and BAKEZYME BXP 5000 and BAKEZYME BXP 5001 (available from DSM).

[0191] The protease may be from Bacillus, e.g., B. amyloliquefaciens. A suitable protease may be Neutrase® available from Novozymes A / S.

[0192] The phospholipase may have phospholipase A1 , A2, B, C, D or lysophospholipase activity; it may or may not have lipase activity. It may be of animal origin, e.g. from pancreas, snake venom or bee venom, or it may be of microbial origin, e.g., from filamentous fungi, yeast or bacteria, such as Aspergillus or Fusarium, e.g., A. niger, A. oryzae or F. oxysporum. A preferred hpase / phosphohpase from Fusarium oxysporum is disclosed in WO 98 / 26057. Also, the variants described in WO 00 / 32758 may be used.

[0193] Suitable phospholipase compositions are LIPOPAN F, LIPOPAN XTRA, and LIPOPAN MAX (available from Novozymes A / S) or PANAMORE GOLDEN and PANAMORE SPRING (available from DSM).

[0194] Preferably, the one or more additional enzyme is added in an amount of 0.01-1 ,000 mg enzyme protein (mgEP) per kg flour, preferably in an amount of 0.01-500 mg enzyme protein (mgEP) per kg flour, even more preferably in an amount of 0.1-100 mg enzyme protein (mgEP) per kg flour.

[0195] Polynucleotides

[0196] The present invention also relates to polynucleotides encoding a polypeptide having alpha-amylase activity of the invention, as described herein. The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof. The polynucleotide may be cloned from a strain of Talaromyces, or a related organism and thus, for example, may be a polynucleotide sequence encoding a variant of the polypeptide of the invention.

[0197] The polynucleotide may also be mutated by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.

[0198] Nucleic Acid Constructs and Expression Vectors

[0199] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a polypeptide of the present invention having alpha-amylase activity, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0200] The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0201] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a polypeptide of the present invention having alpha-amylase activity, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0202] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0203] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0204] The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0205] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0206] For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).

[0207] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0208] More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0209] Host Cells

[0210] The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.

[0211] A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The polypeptide can be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention. Preferably, the host cell comprises at least two copies, e.g., three, four, or five, or more copies of the polynucleotide of the invention or the nucleic acid construct or expression vector of the invention.

[0212] The host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.

[0213] The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Grampositive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coll, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0214] The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus lichen! formis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In an embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus lichen! formis and Bacillus subtilis cell.

[0215] For purposes of this invention, Bacillus classes / genera / species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.

[0216] The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells. The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0217] Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods BA'. 391-397, and Donald et al., 2013, J. Bacteriol. 195(11): 2612- 2620.

[0218] The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0219] Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Christensen et al., 1988, Bio / Technology6'. 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.

[0220] The fungal host cell may be a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). For purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0221] The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell (Komagataella phaffii).

[0222] The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides, vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0223] The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0224] For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0225] In an aspect, the host cell is isolated and / or purified

[0226] Methods of Production

[0227] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide. The present invention also relates to methods of producing a polypeptide having alphaamylase activity, comprising cultivating the recombinant host cell of the invention under conditions conducive for production of the alpha-amylase polypeptide.

[0228] The host cell is cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state, and / or microcarrier-based fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and / or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0229] The polypeptide may be detected using methods known in the art that are specific for the polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an assay determining the relative or specific activity of the polypeptide.

[0230] The polypeptide may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the polypeptide is recovered. In another aspect, a cell-free fermentation broth comprising the polypeptide is recovered.

[0231] The polypeptide may be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science’ 80(1): 6.1 .1-6.1 .35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0232] In an alternative aspect, the polypeptide is not recovered.

[0233] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0234] EXAMPLES

[0235] Example 1 : Talaromyces flavus genomic DNA extraction

[0236] Talaromyces flavus was inoculated onto a PDA plate and incubated for 7 days at 25°C in the darkness. Several mycelia-PDA plugs were inoculated into 500 ml shake flasks containing 100 ml of YPG medium. The flasks were incubated for 3 days at 25°C with shaking at 160 rpm. The mycelia were collected by filtration through MIRACLOTH® (Calbiochem, La Jolla, CA, USA) and frozen under liquid nitrogen. Frozen mycelia were ground, by a mortar and a pestle, to a fine powder, and genomic DNA was isolated using DNeasy® Plant Maxi Kit (24) (QIAGEN GmbH, Hilden, Germany) following the manufacturer’s instructions.

[0237] Example 2: Cloning of the Talaromyces flavus amylase gene from genomic DNA and construction of an Aspergillus Oryzae expression Vector

[0238] The two synthetic oligonucleotide primers shown in table 1 below were designed to PCR amplify the Talaromyces flavus amylase-encoding gene from the genomic DNA prepared in Example 1 . An IN-FUSION CF Dry-down Cloning Kit (Clontech Laboratories, Inc., Mountain View, Calif., USA) was used to clone the fragment directly into the expression vector pPFJO355, without the need for restriction digestion and ligation. The expression vector pPFJO355 contains the Aspergillus Oryzae TAKA alpha-amylase promoter and the Aspergillus niger glucoamylase terminator. Furthermore, pPFJO355 has puC18 derived sequences for selection and propagation in E. coli, and a pyrG gene, which encodes an orotidine decarboxylase for selection of a transformant of a pyrG mutant Aspergillus strain. The expression vector pPFJO355 is described in detail in WO 2011 / 005867.

[0239] Table 1. Bold letters represent coding sequence. The remaining sequence is homologous to the insertion sites of pPFJO355.

[0240] Ten picomoles of each of the primers above were used in a PCR composed of Talaromyces flavus genomic DNA, 10ul of 5xGC Buffer, 1 .5 ul of DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP and 1 unit of PHUSION High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a final volume of 50 pl. The amplification was performed using a Peltier Thermal Cycler programmed for 1 cycle at 98°C for 1 minute; 9 cycles of denaturing at 98°C for 20 seconds, annealing at 60°C for 30 seconds with a 1 ° C increase per cycle, and elongation at 72°C for 2 minutes; and another 25 cycles each at 98°C for 20 seconds, 70° C for 30 seconds and 72° C for 2 minutes; final extension at 72°C for 10 minutes. The heat block then went to 4°C. The resulting reaction products were isolated by 1 .0% agarose gel electrophoresis using TBE buffer where an approximately 2.3 kb product band was excised from the gel, and purified using an illustra GFX PCR DNA and Gel Band Purification Kit according to the manufacturers instructions. Plasmid pPFJO355 was digested with BamHI and Bgl II, isolated by 1.0% agarose gel electrophoresis using TBE buffer, and purified using an illustra GFX PCR DNA and Gel Band Purification Kit according to the manufacturers instructions. The gene fragment and the digested vector were ligated together using an IN-FUSION CF Dry-down PCR Cloning Kit resulting in transcription of the amylase gene was under the control of the Aspergillus Oryzae TAKA alphaamylase promoter. In brief, 30 ng of pPFJO355 digested with Bam HI and Bgl II, and 80 ng purified PCR product were added to a reaction vial and resuspended in a final volume of 10 pl with addition of deionized water. The reaction was incubated at 37° C for 30 minutes and then 50° C for 15 minutes. 3pl of the reaction was used to transform E. coli TOP10 competent cells (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China). An E. coli transformant containing Talaromyces flavus amylase was detected by colony PCR and plasmid DNA was prepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia, Calif., USA). The Talaromyces flavus amylase-encoding gene insertion was further confirmed by DNA sequencing.

[0241] Example 3: Expression of Talaromyces flavus amylase in Aspergillus Oryzae Jal250 strain

[0242] Aspergillus oryzae JaL250 (WO 99 / 61651) protoplasts were prepared according to the method of Christensen et al., 1988, Bio / Technology 6: 1419-1422. 3pg plasmids from Example 2 was transformed into Aspergillus Oryzae Jal250 strain. Six transformants were isolated to a 24- well PDA plate and incubated for 4 days at 30°C. Confluent spores from each well of 24-well plate were washed with 500pl of 0.01% TWEEN® 80 and 20 pl of the spore suspension was used to inoculate 3 ml of YPM medium equipped with 5% maltose in 24-well plate. Transformant cultures were incubated at 30°C with constant shaking at 200 rpm.

[0243] At day four post-inoculation, supernatants were collected. 10pl of each supernatant were mixed with an equal volume of 2X loading buffer (10% beta-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-glycine SDS-PAGE gel and stained with SIMPLY BLUE™ SafeStain (Invitrogen, Carlsbad, CA, USA). SDS-PAGE profiles of the culture broths showed that six out of six transformants had a new protein band of approximately 68 kDa.

[0244] Transformant number 1 was selected and its genomic DNA was isolated. Insertion of the Talaromyces flavus amylase-encoding gene in the Aspergillus Oryzae Jal250 strain genome was further confirmed by spore PCR and DNA sequencing. Transformant number 1 was inoculated onto individual PDA plate and incubated for 4-5 days at 30°C until confluent sporulation. PDA plate was then washed with 2ml of 0.01% TWEEN® 80 and spore suspension was transferred to 2L shake flasks each containing 400ml of YPM supplemented with 5% maltose, shaking at 30°C, 80rpm, 4 days. The culture broths were then harvested by using a 1000ml Rapid-Flow Bottle Top Filter 0.2pm aPES membrane (ThermoFisher Scientific, Cat# 597-4520) and were sent for purification.

[0245] Example 4: Cloning of Talaromyces aculeatus amylase, Talaromyces purpureogenus amylase, Talaromyces calidicanius amylase, Talaromyces pinophilus amylase, Talaromyces sp-71323 amylase and a synthetic fusion amylase in the Aspergillus oryzae Cols1300 strain The Aspergillus oryzae Cols'! 300 strain (US2019225988) was used as the host for heterologous expression of amylase genes by employing the highly efficient so-called doublesplit marker genomic integration system (DSMS). A. oryzae Cols1300 strain (kusA-, niaD-, niiA-) was created from Aspergillus oryzae strain PFJO220 (EP2147107) by deleting the promoter and 5' part of both the nitrite reductase (niiA) and nitrate reductase niaD) genes, thereby inactivating their expression so that an incoming construct flanked with the complementary parts of each gene can be integrated by double homologous recombinations to reconstitute both genes and allow selection of the resulting double integrants on minimal medium with NaNO3as sole nitrogen source (Nielsen M. L. et al. 2006, Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans, Fungal Genetics and Biology vol. 43: 54-64). The A. oryzae Cols1300 strain is capable of assembling several polynucleotide fragments in vivo with each fragment containing as little as a 20 bp overlap with another fragment for successful homologous recombination (EP3512953).

[0246] Genes encoding Talaromyces aculeatus amylase, Talaromyces purpureogenus amylase, Talaromyces calidicanius amylase, Talaromyces pinophilus amylase, Talaromyces sp-71323 amylase and synthetic fusion amylase were codon optimized for Aspergillus oryzae expression using GeneDesigner, a similar codon optimization algorithm to the one described in “Codon bias and heterologous protein expression". Claes Gustafsson, Sridhar Govindarajan and Jeremy Minshull. TRENDS in Biotechnology Vol.22 No.7 July 2004.

[0247] Synthetic DNA fragments were ordered from Azenta Life Sciences (Suzhou, China). The 5’ and 3’ ends of the synthetic fragments contained 22 bp overlaps for homologs recombinations in A. oryzae Cols'! 300.

[0248] The plasmid of expression vector pLYcutsphis derived from pBGMH0016 (WO 2013 / 163590) contains flanking regions that can repair the niiA gene and niaD genes in Cols1300. In this case, 2 fragments, Fragment I (~3.6 kb) and Fragment III (~3.2 kb), were PCR amplified by using primer pairs (dsms0003 & F1 nativesp_R for Fragment I; F3his_F & dsms0004 for Fragment III):

[0249] The coding sequence of the Talaromyces flavus amylase of SEQ ID NO:6 is shown in SEQ ID NO:25.

[0250] The coding sequence of the Talaromyces aculeatus amylase of SEQ ID NO:5 is shown in SEQ ID NO:26.

[0251] The coding sequence of the Talaromyces purpureogenus amylase of SEQ ID NO:4 is shown in SEQ ID NO:27. The coding sequence of the Talaromyces cahdicamus amylase of SEQ ID NO:1 is shown in SEQ ID NO:28.

[0252] The coding sequence of the Talaromyces pinophilus amylase of SEQ ID NO:2 is shown in SEQ ID NO:29.

[0253] The coding sequence of the Talaromyces sp-71323 amylase of SEQ ID NO:3 is shown in SEQ ID NQ:30.

[0254] The coding sequence of the synthetic fusion amylase of SEQ ID NO:7 is shown in SEQ ID NO:31 ; it is a cDNA sequence codon optimized for Aspergillus oryzae expression, containing wild type Fungamyl® backbone with the CBM20 domain from SEQ ID NO: 7 (signal: 1-60; artifical linker: 1495-1557; CBM20:1558-1881).

[0255] Ten microliter of each primer was used in a PCR composed of pLYcutsphis plasmids, 10pl KAPA HiFi Fidelity buffer (5x, Kapa Biosystems), 2.5mM each of dATP, dTTP, dGTP, and dCTP and 1 unit of KAPA HiFi Fidelity Polymerase (Kapa Biosystems) in a final volume of 50pl. The amplification was performed using Bio-Rad C1000 Touch Thermal Cycler for 1 cycle at 98°C for 1 minute; 40 cycles of denaturing at 98°C for 15 seconds, annealing at 68°C for 30 seconds and extension at 72°C for 2 minutes; final extension at 72°C for 10 minutes. The heat block then went to 4°C.

[0256] Ethanol precipitation for purifying and concentrating Fl and Fill fragments after PCR amplification was carried out by following procedures: eight PCR amplification reactions of fragment Fl and Fill were pooled respectively, that makes 400pl sample for each fragment. For each fragment, 0.1 volume (in our case 40pl) of 3M NaAC was added to the pool followed by 2.5 volume (1 OOOpI) of 96% Ethanol. After keeping at -20°C for 30min, samples were centrifuged at 4°C, 8000 rpm for 30min. The pellet was then carefully washed twice with 70% ethanol, 500 pl each time and centrifuge again at 4°C, 13000rpm for 2min. Then, the pellet was dried at room temperature for 5 -10min and resuspended with 40pl ddH2O. The concentrations of fragment Fl and Fill were measured by Qubit™ dsDNA Quantification Assay Kits.

[0257] A. oryzae Cols1300 protoplasts were prepared as in US2019225988. Briefly, the Cols1300 cells were inoculated in 100 ml YPG medium supplemented with 10 mM uridine and incubated for 16 hours at 32°C at 80 rpm. Pellets were collected and washed with 0.6 M KCI and resuspended in 20 ml 0.6 M KCI containing a commercial B-glucanase product (GLUCANEX™, Novozymes A / S, Bagsvaerd, Denmark) at a final concentration of 20 mg per ml. The suspension was incubated at 32°C with shaking (80 rpm) until protoplasts were formed, and then washed twice with STC buffer. The protoplasts were counted, resuspended and adjusted in an 8:2:0.1 solution of STC:STPC:DMSO to a final concentration of 2.5x107protoplasts / ml. Approximately 1 pg of synthesized amylase gene fragments, 500pg of Fl and 500pg of Fill fragments were mixed together with 0.5pl 100mM DTT, and incubated at room temperature for 10 minutes. Then the mixture was transferred to 4X106protoplast and gently mixed. 10OpI 60% PEG solution was added and incubated at room temperature for 30min. Then 50pl STC buffer was added to the mixture and it was transferred to one well of 24-well plate with minimal medium with NaNO3as sole nitrogen source and air dried for 5-10 mins. The 24-well plate was incubated at 30°C for 4- 5 days. Only spores, where the integration cassette had successfully recombined into the chromosome of the host cell and reconstituted the niiA and niaD sites in the process could germinate and survive.

[0258] Four transformants for each amylase molecule were isolated to 24-well plate with NaNO3minimal medium and incubated for 4 days at 30°C. Confluent spores from each well of 24-well plate were washed with 500pl of 0.01% TWEEN® 80 and 200pl of spore suspension was used for genomic DNA isolation. Insertion of each of the Talaromyces aculeatus amylase, Talaromyces purpureogenus amylase, Talaromyces calidicanius amylase, Talaromyces pinophilus amylase, Talaromyces sp 71323 amylase and the synthetic fusion amylase encoding genes in an A. oryzae Cols1300 strain was further confirmed by spore PCR and DNA sequencing. 20 pl of the spore suspension from one sequence verified transformant for each amylase was used to inoculate 3 ml of YPM medium supplemented with 5% maltose in 24-well plate. Transformant cultures were incubated at 30°C with constant shaking at 200 rpm. At day four post-inoculation, supernatants were collected. 15pl of each supernatant were mixed with 5pl sample buffer (NuPAGE™ LDS Sample Buffer, 4X) and loaded onto a 1.5 mm 8%-16% Tris-glycine SDS-PAGE gel. The SDS- PAGE gels were then stained with InstantBlue™ Protein Stain (Sigma-Aldrich). SDS-PAGE profiles of all the culture broths showed a new protein band of approximately 66 kDa.

[0259] Single colonies were used to inoculate slants with NaNO3minimal medium. When they were fully sporulated, spores were collected in 2ml of YPM medium and transferred to 2L shake flasks each containing 400ml of YPM supplemented with 5% maltose, shaking at 30°C, 80rpm, 4 days. The culture broths were then harvested using a 1000ml Rapid-Flow Bottle Top Filter 0.2pm aPES membrane (ThermoFisher Scientific, Cat# 597-4520) and were sent for purification.

[0260] Example 5: Purification of amylase from Aspergillus oryzae transformants

[0261] Fermentation broths (800ml) of each of the amylases is filtered by 0.22 pm filter (Millex- HV, Millipore), then the conductance of the filtered fermentation broth is adjusted to 175-180 mS / cm by adding 4 M ammonium sulfate using a conductivity meter (SevenEasy Conductivity, Mettler Toledo AG). The solutions containing the target amylase is loaded separately onto a HIC column (phenyl sepharose high performance , Cytiva) equilibrated with 2 column volumes (CV) 0.1 M NaOH, 2 CV ddH2O, 2 CV 20mm PBS pH7.5, 1 ,9M (NH4)2SO4sequentially.

[0262] To remove unbound materials, the column is washed with 2 CV 20mm PBS pH7.5, (NH4)2SO4. Amylases is eluted by a linear (NH4)2SO4gradients (from 1 ,9M to 0 mM (NH4)2SO4in 14 CV) and fractions containing the target protein are selected and pooled based on SDS- PAGE (NUPAGE NOVEX 4-12% Bis-Tris Gel, NoVex) results. Eluted amylases are concentrated by a 10 kDa concentrator (VIVASPIN 20, Sartorius), the purity and concertation of the protein were determined by SDS-PAGE. Example 6: Thermal stability test of amylases of the invention by nano DSF

[0263] Each purified recombinant amylase was diluted by 50 mM pH buffer (MES, Glycine, Sodium acetate, pH 3-7) to make a 100 pl protein solution in triplicate, with protein concentrations between 0.1 mg / ml~0.2 mg / ml. Diluted protein solutions were loaded to NanoDSF(Prometheus NT.Plex, NanoTemper) with a linear temperature ramp 20-95°C at 3.3°C / min. The intrinsic fluorescence ratios of F350 / F330 were plotted against temperature, inflection points of the melting curve were determined by PR.ThermControl software(NanoTemper). Results are shown in table 2 below.

[0264] Table 2.

[0265] Example 7: Impact of amylases of the invention on bread volume

[0266] Fungamyl® is often used in baking applications to improve baking performance. The improved baking performance can be seen as an increase in specific volume and decrease in firmness of the bread crumb as well as impacting the dough properties and crust color. In this trial an improved amylase was tested compared to the benchmark, Fungamyl®. The breads were prepared in a straight dough process with a recipe according to

[0267] Table3 below. Different treatments were made according to Table4. The breads were baked as free-standing rolls to determine the impact on volume of the final bread.

[0268] Table 3: Recipe

[0269] Table 4: Treatments, each enzyme and dosage were run in duplicates in a randomized order The ingredients and different treatments were placed in a pin mixer and mixed into a dough for 4 min at 90 rpm. The dough was taken out from the mixer and allowed to rest for 5 minutes.

[0270] The dough was divided into 18 g dough pieces. The dough pieces were rounded and placed in a proofing cabinet. The doughs were proofed for 80 min at 30°C and 80% relative humidity.

[0271] The proofed doughs were placed on a conveyor belt and baked in a continuous oven for 12 min at 210 °C.

[0272] After passing through the oven on the conveyor belt the bread passed through a laser equipped volume scanner (Videometer, Denmark) that determined the volume based on three pictures taken from different angles and a balance where the weight of the bread was determined.

[0273] The specific volume (ml / g) was calculated by dividing the volume by the weight.

[0274] The addition of Fungamyl® increased the volume of the bread as can be seen in Table5. The higher the dosage the bigger the increase on the specific volume of the bread.

[0275] The addition of any of the tested amylases was even more efficient in increasing the volume of the bread as can be seen in Table5. A dosage of 0.5 mgEP / kg flour of the novel amylases increased the specific volume more than 0.5 mgEP / kg flour of Fungamyl®. A dosage of 1 .25 mgEP / kg flour of the novel amylases increased the specific volume more than 1 .25 mgEP / kg flour of Fungamyl®.

[0276] In fact, a dosage of 1 ,25 mgEP of the novel amylases increased the specific volume to the same degree or more than a 2.5 mgEP / kg flour dosage of Fungamyl®.

[0277] Example 8: Impact of amylases of the invention on bread volume

[0278] Fungamyl® is often used in baking applications to improve baking performance. The improved baking performance can be seen as an increase in specific volume and decrease in firmness of the bread crumb as well as impacting the dough properties and crust color. In this trial an improved amylase was tested compared to the benchmark, Fungamyl®. The breads were prepared in a straight dough process with a recipe according to Table 6. Different treatments were made according to Table 7. The bread was baked as free-standing rolls to determine the impact on volume of the final bread.

[0279] Table 6: Recipe

[0280] Table 7: Treatments, each enzyme and dosage were run in duplicates in a randomized order

[0281] The ingredients and different treatments were placed in a pin mixer and mixed into a dough for 4 min at 90 rpm. The dough was taken out from the mixer and allowed to rest for 5 minutes.

[0282] The dough was divided into 18 g dough pieces. The dough pieces were rounded and placed in a proofing cabinet. The doughs were proofed for 80 min at 30°C and 80% relative humidity.

[0283] The proofed doughs were placed on a conveyor belt and baked in a continuous oven for 12 min at 210°C.

[0284] After passing through the oven on the conveyor belt the bread passed through a laser equipped volume scanner (Videometer, Denmark) that determined the volume based on three pictures taken from different angles and a balance where the weight of the bread was determined. The specific volume (ml / g) was calculated by dividing the volume by the weight. Table 8: Specific volumes of baked bread vs. enzyme dosages

[0285] The addition of Fungamyl® increased the volume of the bread as can be seen in Table 8. The higher the dosage the bigger the increase on specific volume of the bread.

[0286] The addition of any of the tested amylases was even more efficient in increasing the volume of the bread as can be seen in Table 8. A dosage of 0.5 mgEP / kg flour of the novel amylases increased the specific volume more than 0.5 mgEP / kg flour of Fungamyl®. A dosage of 1 .25 mgEP / kg flour of the novel amylases increased the specific volume more than 1 .25 mgEP / kg flour of Fungamyl®.

[0287] In fact, a dosage of 1 ,25 mgEP of the novel amylases increase the specific volume to the same degree or more than a 2.5 mgEP / kg flour dosage of Fungamyl®.

[0288] Example 9: Impact of amylases of the invention on bread volume

[0289] Fungamyl® is often used in baking applications to improve baking performance. The improved baking performance can be seen as an increase in specific volume and decrease in firmness of the bread crumb as well as impacting the dough properties and crust color. In this trial an improved amylase was tested compared to the benchmark, Fungamyl®. The breads were baked in a straight dough baking process with a recipe according to

[0290] Table9. Different treatments were made according to Table 10 The bread was baked in open tins (steel plate and steel ring) to determine the impact on volume of the final bread.

[0291] Table 9: Recipe

[0292] Table 10: Treatments, each enzyme and dosage were run in duplicates in a randomized order

[0293] The ingredients and different treatments were placed in a pin mixer and mixed into a dough for 4 min at 90 rpm. The dough was taken out from the mixer and allowed to rest for 5 minutes The dough was divided into 18 g dough pieces. The dough pieces were rounded and placed on a steel plate surrounded by a 55 mm steel ring. The dough in the steel ring was placed on a conveyor belt that was running through a continuous proofing tunnel. The speed of the conveyor belt was set so the buns were proofed for 55 min at 36 °C and 85% relative humidity.

[0294] The proofed doughs transferred to a conveyor belt running through a continuous oven and baked the buns for 12 min at 210 °C.

[0295] After passing through the oven, on the conveyor belt the bread passed through a laser equipped volume scanner (Videometer, Denmark) that determined the volume based on three pictures taken from different angles and a balance where the weight of the bread was determined. The specific volume (ml / g) is calculated by dividing the volume by weight.

[0296] Table 11 : Volume of bread

[0297] The addition of Fungamyl® increased the volume of the bread as can be seen in table 11 above. The addition of any of the other tested amylases was even more efficient in increasing the volume of the bread as can be seen in Table 11. The novel amylases increased the specific volume to the same extent or more at a dosage of 2.5 mgEP / kg flour compared to a 5 mgEP / kg flour dosage of Fungamyl®.

[0298] Example 10: Impact of amylase of the invention on bread volume

[0299] Fungamyl® is often used in baking applications to improve baking performance. The improved baking performance can be seen as an increase in specific volume and decrease in firmness of the bread crumb. In this trial an improved amylase was tested compared to the benchmark, Fungamyl®. The Breads were baked in a straight dough baking process with a recipe according to

[0300] Table12. Different treatments were made according to Table13. The bread was baked in open tins to determine the impact on volume of the final bread.

[0301] Table 12: Recipe

[0302] Table 13: Treatments, each enzyme and dosage were run in duplicates in a randomized order

[0303] The ingredients and different treatments were placed in a spiral mixer and mixed into a dough for 3 min at 17 rpm and 7 min at 35 rpm. The dough was taken out from the mixer and allowed to rest for 5 minutes

[0304] The dough properties were determined by a trained baker. The doughs were evaluated on 4 parameters relative to the blank on stickiness, softness, extensibility, and elasticity on a scale from 1 to 9. The blank was given a score of 5 and the other doughs were given a score relative to the blank. A higher intensity of the parameter was given a higher score, and a lower intensity was given a lower score. A small difference was given one score away from the blank while a clear difference was given 2 scores away from the blank. Even bigger differences compared to the blank was given even more extreme scores. The properties stickiness, softness, extensibility, and elasticity were determined.

[0305] The dough was divided into 320 g dough pieces. The dough pieces were rounded and allowed to rest for 20 minutes. After the rest, the dough pieces were sheeted and placed in baking tins. The tins with the doughs were proofed for 60 and 85 min at 32°C and 86% relative humidity, normal and extended proofing, respectively.

[0306] The proofed doughs were baked in a deck oven for 28 min at 230 °C.

[0307] The breads were allowed to cool down and volume of the was determined 2 hours after baking. The volume of the bread was determined using a benchtop laser-based contour scanner that measured the volume, density, and dimensional profiles of solid products (Volscan Profile, Stable Micro Systems, Godaiming, UK). A standard method for measuring the volume of a bread is to tare the balance of the instrument and calibrate it to zero height. Place the bread on a plate with supporting pins. Start a scan with 1 ,5 rpm and 26 mm vertical step. The weight (gram) is determined by the in-built balance, the volume (ml) is determined from the scanned contour of the bread and the specific volume (ml / g) is calculated by dividing the volume by the weight.

[0308] The bread was packed 2 hours after baking in sealed plastic bags and stored at room temperature until texture analysis.

[0309] The texture of the bread was evaluated with a texture analyzer (TA-XT plus, Stable microsystems, Godalmine, UK). Bread crumb texture properties were characterized by firmness (the same as “hardness” and the opposite of “softness”) and the elasticity of the baked product. A standard method for measuring firmness and elasticity is based on force-deformation of the baked product. A force-deformation of the baked products may be performed with a 40 mm diameter cylindrical probe. The force on the cylindrical probe is recorded as it is pressed down to a 40% strain on a 25 mm thick bread slice at a deformation speed of 1 mm / second. The probe is then kept in this position for 30 seconds while the force is recorded and then probe returns to its original position.

[0310] Firmness (in grams) is defined as the force needed to compress a probe to a 25% strain (corresponding to 6,25 mm compression into a bread crumb slice of 25 mm thickness).

[0311] Elasticity (in %) is defined as the force recoded after 30 seconds compression at 40% strain (corresponding to force at time=40s for a bread slice of 25 mm thickness) divided by the force needed to press the probe 10 mm into the crumb (corresponding to force at time=10 s for a bread slice of 25 mm thickness) times 100.

[0312] Table 14: Specific volume of baked bread vs. enzyme dosage

[0313] The addition of Fungamyl® increased the volume of the bread using both normal and extended proofing time as can be seen in Table14. The higher the dosage the bigger the increase on specific volume of the bread. The Firmness of the bread crumb decreased with the addition of Fungamyl® while the elasticity of the bread crumb was not affected. As can be seen in Table5.

[0314] The addition of the amylase with the amino acid sequence shown in SEQ ID NO:6 was even more efficient in increasing the volume of the bread as well as decreasing the firmness of the bread crumb as can be seen in Tablei 4 and Table5. A dosage of 1 mgEP / kg flour of this amylase increased the specific volume and lowered the Firmness more than 1 mgEP / kg flour of Fungamyl®, which is a desirable result.

[0315] In fact, a dosage of 1 ,5 mgEP of the amylase of SEQ ID NO:6 increased the specific volume more than 5 mgEP / kg flour of Fungamyl® while lowering the firmness to the same degree. Example 11 : Impact of amylase of the invention on bread volume

[0316] Fungamyl® is often used in baking applications to improve baking performance. The improved baking performance can be seen as an increase in specific volume and decrease in firmness of the bread crumb. In this trial five improved amylases were tested compared to the benchmark, Fungamyl®. The trial was done in a straight dough process as described in example 10 with the ingredients listed in the recipe table 16 below, and the different amylase enzyme treatments listed in Table 27. Table 16: Recipe

[0317] Table 27: Treatments, each enzyme and dosage were run in duplicate in a randomized order

[0318] Table 38: Volume of bread

[0319]

[0320] Table 49: Firmness and Elasticity of bread crumb on day 1 after baking The addition of Fungamyl® increased the volume of the bread using both normal and extended proofing time as can be seen in Table 38. The higherthe dosage the biggerthe increase on specific volume of the bread. The Firmness of the bread crumb decreased with the addition of Fungamyl® while the elasticity of the bread crumb was not affected, as can be seen in the above table 19Table.

[0321] The addition of any of the amylases of the invention was more efficient at increasing the volume of the bread than the benchmark, Fungamyl®, as well as at decreasing the Firmness of the bread crumb, both of which are commercially desirable results, as can be seen in Table 38 and Table 49. A dosage of 0,5 mgEP / kg flour of any of the amylases listed increased the specific volume and lowered the Firmness more than 0,5 mgEP / kg flour of Fungamyl®.

[0322] In fact, a dosage as low as 0,2 mgEP of any of the amylases of the invention increased the specific volume more than 0,5 mgEP / kg flour of Fungamyl® while lowering the Firmness to a similar extent as the higher dosed Fungamyl®.

[0323] Example 12: Impact of the amylase of the invention with xylanase on bread volume

[0324] Fungamyl® is often used in baking applications in combination with a xylanase to improve the baking performance. The improved baking performance can be seen as an increase in specific volume and decrease in firmness of the bread crumb. In this trial an improved amylase was tested together with different commercially available xylanases typically used in baking applications and compared to the benchmark, Fungamyl®, in combination with the same xylanases. The trial was run in a straight dough process as described in example 10.

[0325] Table 20: Recipe

[0326] Table 21 : Treatments, each treatment was run in duplicate in a randomized order

[0327]

[0328] Table 22: Specific volume of bread vs enzymes

[0329] Table 23: Firmness and Elasticity of bread crumb on day 1 after baking

[0330] The addition of Fungamyl® increased the volume of the bread using both normal and extended proofing time as can be seen in Table22. The Firmness of the bread crumb decreased with the addition of Fungamyl® as can be seen in Table23Table.

[0331] The addition of 0.625 mgEP / kg flour of the amylase with the amino acid sequence showed in SEQ ID NO:6 increased the specific volume even more than 2.5 mgPE / kg flour of Fungamyl® and also lowered the firmness of the bread crumb.

[0332] The addition of either of the xylanases Panzea®, Pentopan® Mono or Pentopan® 500 increased the volume of the bread using both normal and extended proofing time as can be seen in Table22 and at the same time lowered the firmness as can be seen in Table23Table.

[0333] The combination of the benchmark and any of the xylanases increased the specific volume further than any of the enzymes alone.

[0334] The combination of 0.625mgEP / kg flour of the amylase having the amino acid sequence shown in SEQ ID NO:6 in combination of any of the commercially available xylanases listed above increased the specific volume to the same extent as 2,5mgEP / kg of flour in combination with any of the xylanases.

[0335] Example 13: Impact of carbohydrate binding modulus on the performance of the amylase of the invention on bread volume

[0336] Fungamyl® is often used in baking applications to improve baking performance. The improved baking performance can be seen as an increase in specific volume and decrease in firmness of the bread crumb as well as impacting the dough properties and crust color. In this trial an improved amylase was tested in 2 versions: 1) Native enzyme with catalytic domain, linker and carbohydrate modulus (CBM) as shown in SEQ ID NO:5; and 2) A truncated version of SEQ ID NO:5 containing the same catalytic domain but without its linker and CBM, i.e. “SEQ ID NO:5 - CBM”; the truncated version consisted of the amino acid sequence shown in positions 20-498 of SEQ ID NO:5. The breads were prepared in a straight dough process with a recipe according to Table 24 below. Different treatments were made according to Table 25. The bread was proofed at two different proofing times and baked in open bread tins to determine the impact on volume of the final bread.

[0337] The concentration of the two samples was determined by amino acid analysis, and the molecular weight (Mw) by intact mass analysis, both using mass spectrometry. From this both the molecular weight (Da) and concentration were determined. The Mw of the native amylase was 66.5 kDa, while the Mw of the catalytic domain alone was 52.7 kDa. To compare the two samples on equal number of molecules (molars) the concentrations were normalized to native amylase, meaning the determined concentration of SEQ ID NO:5 - CBM was multiplied by 1 .262 (66.5 / 52.7 =1.262).

[0338] Table 24: Recipe

[0339] Table 25: Treatments, each enzyme and dosage were run in duplicates in a randomized order

[0340] The ingredients and different treatments were placed in a spiral mixer and mixed into a dough for 3 min at 17 rpm and 7 min at 35 rpm. The dough was taken out from the mixer and allowed to rest for 15 minutes.

[0341] The dough was divided into 320 g dough pieces. The dough pieces were rounded and allowed to rest for 15 minutes. After the rest, the dough pieces were sheeted and placed in baking tins. The tins with the doughs were proofed for 60 and 85 min at 32°C and 86% relative humidity, normal and extended proofing, respectively.

[0342] The proofed doughs were baked in a deck oven for 28 min at 230 °C. The breads were allowed to cool down and volume of the breads was determined 2 hours after baking. The volume of the bread was determined using a benchtop laser-based contour scanner that measured the volume, density, and dimensional profiles of solid products (Volscan Profile, Stable Micro Systems, Godaiming, UK). A standard method for measuring the volume of a bread is to tare the balance of the instrument and calibrate it to zero height. Place the bread on a plate with supporting pins. Start a scan with 1 ,5 rpm and 26 mm vertical step. The weight (gram) is determined by the in-built balance, the volume (ml) is determined from the scanned contour of the bread and the specific volume (ml / g) is calculated by dividing the volume by the weight.

[0343] Table 26: Specific volume of baked bread vs. enzyme dosage

[0344] The addition of the amylases increased the volume of the bread using both normal and extended proofing time as can be seen in Table 14. The higher the dosage the bigger the increase on specific volume of the bread.

[0345] The addition of the Native enzyme with catalytic domain, linker and carbohydrate modulus (CBM) with the amino acid sequence shown in SEQ ID NO:5 was more efficient compared to the catalytic domain alone in increasing the volume of the bread in table 14. A dosage of 0.25 mgEP / kg flour of the native amylase increased the specific volume more compared to 0.25 mgEP / kg flour of the catalytic domain alone.

[0346] In fact, a dosage of 0.25 mgEP of the native amylase SEQ ID NO:5 increased the specific volume more than 0.5 mgEP / kg flour of the catalytic domain alone. This illustrated the positive effect of adding a linker and carbohydrate binding modulus to the amylase. The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

[0347] The invention is further defined by the following numbered paragraphs:

[0348] 1. A polypeptide having alpha-amylase activity, wherein said polypeptide is selected from the group consisting of:

[0349] (a) a polypeptide having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:4, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:5, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:6, at least 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:9, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 13, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 14, or at least 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:15;

[0350] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1 -30 alterations, e.g. , substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0351] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0352] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0353] 2. A polypeptide having alpha-amylase activity, which is:

[0354] (a) a polypeptide having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:4; or (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1 -30 alterations, e.g. , substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0355] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0356] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity

[0357] 3. A polypeptide having alpha-amylase activity, which is:

[0358] (a) a polypeptide having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:5; or

[0359] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0360] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0361] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0362] 4. A polypeptide having alpha-amylase activity, which is:

[0363] (a) a polypeptide having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:6; or

[0364] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0365] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0366] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0367] 5. A polypeptide having alpha-amylase activity, which is:

[0368] (a) a polypeptide having at least 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:9; or

[0369] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0370] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0371] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0372] 6. A polypeptide having alpha-amylase activity, which is:

[0373] (a) a polypeptide having at least 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 13; or

[0374] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0375] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more ammo acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0376] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0377] 7. A polypeptide having alpha-amylase activity, which is:

[0378] (a) a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:14; or

[0379] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0380] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and

[0381] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0382] 8. A polypeptide having alpha-amylase activity, which is:

[0383] (a) a polypeptide having at least 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:15; or

[0384] (b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0385] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0386] 9. The polypeptide of any one of paragraphs 1-8, comprising, consisting essentially of, or consisting of the mature part of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0387] 10. A polypeptide comprising a catalytic domain selected from the group consisting of:

[0388] (a) a catalytic domain having at least having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:4, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:5, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:6, at least 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:9, at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:13, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO: 14, or at least 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:15;

[0389] (b) a catalytic domain derived from any catalytic domain recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0390] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0391] (d) a fragment of any catalytic domain recited in (a), (b), or (c), wherein the fragment has alpha-amylase activity.

[0392] 11. The polypeptide of paragraph 10, further comprising a native or heterologous carbohydrate binding module, preferably a native or heterologous carbohydrate binding module of CaZy Family 20.

[0393] 12. The polypeptide of any one of paragrahps 1-11 , which is isolated or purified. 13. A granule, which comprises:

[0394] (a) a core comprising the polypeptide of any one of paragraphs 1-12 and optionally,

[0395] (b) a coating consisting of one or more layer(s) surrounding the core.

[0396] 14. A granule, which comprises:

[0397] (a) a core, and

[0398] (b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the polypeptide of any one of paragraphs 1-12.

[0399] 15. A liquid composition comprising the polypeptide of any one of paragraphs 1-12 and an enzyme stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).

[0400] 16. The liquid composition of paragraph 15, further comprising a filler or carrier material.

[0401] 17. The liquid composition of paragraph 15 or 16, further comprising a preservative.

[0402] 18. A composition comprising the polypeptide of any one of paragraphs 1-12, the granule of paragraph 13 or 14, or the liquid composition of any one of paragraphs 15-17.

[0403] 19. The composition of paragraph 18, which is a liquid composition, solid composition, solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry, gel or pellet.

[0404] 20. A baking composition comprising a baking ingredient and a polypeptide having alphaamylase activity, wherein said polypeptide is selected from the group consisting of:

[0405] (a) a polypeptide having at least 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:1 , a polypeptide having at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:2, a polypeptide having at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:3, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:4, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:5, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:6, a polypeptide having at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:7, a polypeptide having at least 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:8, a polypeptide having at least 89%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:9, a polypeptide having at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 10, a polypeptide having at least 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:11 , a polypeptide having at least 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 12, a polypeptide having at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:13, a polypeptide having at least 75%, 80%, 85%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:14, a polypeptide having at least 75%, 80%, 85%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:15, a polypeptide having at least 75%, 80%, 85%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:16, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO: 17, a polypeptide having at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the mature sequence of SEQ ID NO:18,

[0406] (b) a polypeptide derived from the polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0407] (c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0408] (d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

[0409] 21. A baking composition comprising a baking ingredient and a polypeptide comprising a catalytic domain having alpha-amylase activity, wherein said catalytic domain is selected from the group consisting of:

[0410] (a) a catalytic domain having at least 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:1 , at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:2 at least 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:3, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:4, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:5, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:6, at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:7, at least 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:8, at least 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:9, at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO: 10, at least 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:11 , at least 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:12, at least 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:13, at least 75%, 80%, 85% 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:14, at least 75%, 80%, 85% 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:15, at least 75%, 80%, 85% 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:16, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:17, at least 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:18,

[0411] (b) a catalytic domain derived from any of the catalytic domains recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;

[0412] (c) a catalytic domain derived from any catalytic domain recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and

[0413] (d) a fragment of any catalytic domain recited in (a), (b), or (c), wherein the catalytic domain fragment retains its alpha-amylase activity. 22. The baking composition of paragraph 21 , wherein the polypeptide comprising a catalytic domain having alpha-amylase activity further comprises a native or heterologous carbohydrate binding module, preferably a native or heterologous carbohydrate binding module of CaZy Family 20.

[0414] 23. The baking composition of any of paragraph 20 or 21 , wherein the baking ingredient comprises flour, fat and / or a leavening agent.

[0415] 24. The baking composition of any of paragraph 20-23, which further comprises an additional enzyme selected from maltogenic alpha-amylase, raw starch degrading alpha-amylase, betaamylase, xylanase, lipase, phospholipase, glucoamylase, protease, cellulase,

[0416] 25. A process for preparing a dough, or optionally a par-baked or baked product prepared from the dough, said process comprising incorporating into the dough a polypeptide as defined in any one of paragraphs 1-12 or a baking composition as defined in any of paragraphs 20-24 to prepare the dough, and optionally par-baking or baking the dough to produce a par-baked or baked product, respectively.

[0417] 26. Use of a polypeptide having alpha-amylase activity as defined in any one of paragraphs 1-12 in a method for producing a dough, a par-baked or a baked product.

[0418] 27. A polynucleotide encoding a polypeptide having alpha-amylase activity as defined in any one of paragraphs 1-22.

[0419] 28. A nucleic acid construct or expression vector comprising the polynucleotide of paragraph 27, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0420] 29. A recombinant host cell comprising the polynucleotide of paragraph 27 or the nucleic acid construct or expression vector of paragraph 28, wherein the polypeptide is produced and, preferably, secreted.

[0421] 30. The recombinant host cell of paragraph 29, wherein the polypeptide is heterologous to the recombinant host cell.

[0422] 31 . The recombinant host cell of paragraph 29 or 30, wherein at least one of the one or more control sequences is heterologous to the polynucleotide encoding the polypeptide. 32. The recombinant host cell of any one of paragraphs 29-31 , which comprises at least two copies, e.g., three, four, or five, or more copies of the polynucleotide of paragraph 27 or the nucleic acid construct or expression vector of paragraph 28.

[0423] 33. The recombinant host cell of any one of paragraphs 29-32, which is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0424] 34. The recombinant host cell of any one of paragraphs 29-32, which is a filamentous fungal recombinant host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0425] 35. The recombinant host cell of any one of paragraphs 29-32, which is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coh, Flavobactenum, Fusobactenum, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitiHs, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces Hvidans cells.

[0426] 36. The recombinant host cell of any one of paragraphs 29-35, which is isolated.

[0427] 37. The recombinant host cell of any one of paragraphs 29-36, which is purified.

[0428] 38. A method of producing the polypeptide having alpha-amylase activity as defined in any one of paragraphs 1-22, comprising cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide.

[0429] 39. The method of paragraph 38, further comprising recovering and / or purifying the polypeptide.

[0430] 40. A method of producing a polypeptide having alpha-amylase activity, comprising cultivating the recombinant host cell of any one of paragraphs 29-36 under conditions conducive for production of the polypeptide.

[0431] 41. The method of paragraph 40, further comprising recovering and / or purifying the polypeptide.

Claims

Claims1. A polypeptide having alpha-amylase activity, wherein said polypeptide is selected from the group consisting of:(a) a polypeptide having at least 92% sequence identity to the mature sequence of SEQ ID NO:4, at least 92% sequence identity to the mature sequence of SEQ ID NO:5, at least 92% sequence identity to the mature sequence of SEQ ID NO:6, at least 94% sequence identity to the mature sequence of SEQ ID NO:9, at least 92% sequence identity to the mature sequence of SEQ ID NO: 13, at least 75% sequence identity to the mature sequence of SEQ ID NO: 14, or at least 97% sequence identity to the mature sequence of SEQ ID NO: 15;(b) a polypeptide derived from any polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1 -30 alterations, e.g. , substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;(c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids, prefeably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations; and(d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

2. The polypeptide of claim 1 , wherein the polypeptide has at least 95% sequence identity to the mature sequence of SEQ ID NO:4, at least 95% sequence identity to the mature sequence of SEQ ID NO:5, at least 95% sequence identity to the mature sequence of SEQ ID NO:6, at least 95% sequence identity to the mature sequence of SEQ ID NO:9, at least 95% sequence identity to the mature sequence of SEQ ID NO: 13, at least 95% sequence identity to the mature sequence of SEQ ID NO: 14, or at least 98% sequence identity to the mature sequence of SEQ ID NO:15.

3. The polypeptide of any one of claims 1-2, comprising, consisting essentially of, or consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

4. The polypeptide of any one of claims 1-5, comprising, consisting essentially of, or consisting of the mature sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

5. A polypeptide comprising a catalytic domain selected from the group consisting of:(a) a catalytic domain having at least having at least 92% sequence identity to theGH13 alpha-amylase catalytic domain of SEQ ID NO:4, at least 92% sequence identity to theGH13 alpha-amylase catalytic domain of SEQ ID NO:5, at least 92% sequence identity to theGH13 alpha-amylase catalytic domain of SEQ ID NO:6, at least 94% sequence identity to theGH13 alpha-amylase catalytic domain of SEQ ID NO:9, at least 92% sequence identity to theGH13 alpha-amylase catalytic domain of SEQ ID NO:13, at least 75% sequence identity to theGH13 alpha-amylase catalytic domain of SEQ ID NO: 14, or at least 97% sequence identity to theGH13 alpha-amylase catalytic domain of SEQ ID NO:15;(b) a catalytic domain derived from any catalytic domain recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;(c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and(d) a fragment of any catalytic domain recited in (a), (b), or (c), wherein the fragment has alpha-amylase activity.

6. The polypeptide of claim 7, further comprising a native or heterologous carbohydrate binding module, preferably a native or heterologous carbohydrate binding module of CaZy Family 20.

7. A baking composition comprising a baking ingredient and a polypeptide having alphaamylase activity, wherein said polypeptide is selected from the group consisting of:(a) a polypeptide having at least 88% sequence identity to the mature sequence of SEQ ID NO:1 , a polypeptide having at least 87% sequence identity to the mature sequence ofSEQ ID NO:2, a polypeptide having at least 87% sequence identity to the mature sequence ofSEQ ID NO:3, a polypeptide having at least 90% sequence identity to the mature sequence ofSEQ ID NO:4, a polypeptide having at least 90% sequence identity to the mature sequence ofSEQ ID NO:5, a polypeptide having at least 90% sequence identity to the mature sequence ofSEQ ID NO:6, a polypeptide having at least 91% sequence identity to the mature sequence ofSEQ ID NO:7, a polypeptide having at least 88% sequence identity to the mature sequence ofSEQ ID NO:8, a polypeptide having at least 89% sequence identity to the mature sequence ofSEQ ID NO:9, a polypeptide having at least 91% sequence identity to the mature sequence ofSEQ ID NO: 10, a polypeptide having at least 99% sequence identity to the mature sequence ofSEQ ID NO:11 , a polypeptide having at least 99% sequence identity to the mature sequence of SEQ ID NO: 12, a polypeptide having at least 91% sequence identity to the mature sequence ofSEQ ID NO: 13, a polypeptide having at least 75% sequence identity to the mature sequence ofSEQ ID NO: 14, a polypeptide having at least 75% sequence identity to the mature sequence ofSEQ ID NO: 15, a polypeptide having at least 75% sequence identity to the mature sequence ofSEQ ID NO: 16, a polypeptide having at least 90% sequence identity to the mature sequence ofSEQ ID NO: 17, a polypeptide having at least 90% sequence identity to the mature sequence ofSEQ ID NO:18,(b) a polypeptide derived from the polypeptide recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;(c) a polypeptide derived from any polypeptide recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and(d) a fragment of any polypeptide recited in (a), (b), or (c), wherein said polypeptide fragment has alpha-amylase activity.

8. A baking composition comprising a baking ingredient and a polypeptide comprising a catalytic domain having alpha-amylase activity, wherein said catalytic domain is selected from the group consisting of:(a) a catalytic domain having at least 88% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:1 , at least 87% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:2 at least 87% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:3, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:4, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:5, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:6, at least 91% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:7, at least 88% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:8, at least 89% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:9, at least 91% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NQ:10, at least 99% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:11 , at least 99% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:12, at least 91% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:13, at least 75% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:14, at least 75% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:15, at least 75% sequence identity to the GH13 alpha-amylase catalytic domain of SEQ ID NO:16, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO:17, at least 90% sequence identity to the GH13 alphaamylase catalytic domain of SEQ ID NO: 18,(b) a catalytic domain derived from any of the catalytic domains recited in (a) by substitution, deletion or addition of one or several amino acids, preferably by having 1-30 alterations, preferably substitutions, deletions and / or insertions, at one or more positions, preferably 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations;(c) a catalytic domain derived from any catalytic domain recited in (a) or (b), wherein the N- and / or C-terminal end has been extended by addition or shortened by removal of one or more amino acids; and(d) a fragment of any catalytic domain recited in (a), (b), or (c), wherein the catalytic domain fragment retains its alpha-amylase activity.

9. The baking composition of claim 8, wherein the polypeptide comprising a catalytic domain having alpha-amylase activity further comprises a native or heterologous carbohydrate binding module, preferably a native or heterologous carbohydrate binding module of CaZy Family 20.

10. A process for preparing a dough, or optionally a par-baked or baked product prepared from the dough, said process comprising incorporating into the dough a polypeptide as defined in any one of claims 1-6 or a baking composition as defined in any of claims 7-9 to prepare the dough, and optionally par-baking or baking the dough to produce a par-baked or baked product, respectively.

11. Use of a polypeptide having alpha-amylase activity as defined in any one of claims 1-10 in a method for producing a dough, a par-baked or a baked product.

12. A polynucleotide encoding a polypeptide having alpha-amylase activity as defined in any one of claims 1-10.

13. A nucleic acid construct or expression vector comprising the polynucleotide of claim 12, operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

14. A recombinant host cell comprising the nucleic acid construct or expression vector of claim 13.

15. A method of producing a polypeptide having alpha-amylase activity, comprising cultivating the recombinant host cell of claim 14 under conditions conducive for production of the polypeptide and, optionally, recovering the polypeptide.

Images