Variant polypeptide having alpha-amylase activity

Abstract

The present invention relates to novel variant polypeptide exhibiting alpha-amylase activity, the nucleic acid sequence encoding said variant, a recombinant expression vector thereof, and a recombinant host cell containing said expression vector. The invention further extends to a composition comprising the alpha-amylase variant polypeptide of the invention, its use for the preparation of baked products, and a procedure for producing said baked products.

Description

VARIANT POLYPEPTIDE HAVING ALPHA- AMYLASE ACTIVITY FIELD OF THE INVENTION

[0001] The present invention pertains to a novel variant polypeptide exhibiting alpha-amylase activity. Additionally, it encompasses a nucleic acid sequence responsible for encoding said variant, a recombinant expression vector thereof, and a recombinant host cell containing said expression vector. Moreover, the invention entails a methodology for the production of alpha-amylase utilizing said host cell. Furthermore, the present invention encompasses a method for generating a variant of the alpha-amylase polypeptide. The invention further extends to a composition comprising the alpha-amylase variant, its utilization or the utilization of a composition containing such a variant in the creation of baked products, and a procedure for producing said baked products.BACKGROUND OF THE INVENTION

[0002] Alpha-amylases represent a diverse class of enzymes crucial in various biological and industrial processes. Classified into distinct types, including bacterial, fungal, and plant-derived variants, alphaamylases exhibit remarkable versatility in their substrate specificity and catalytic activity. Bacterial alpha-amylases, such as those from Bacillus species, are renowned for their robustness and thermostability, making them invaluable in industrial applications such as starch liquefaction and saccharification for bioethanol production. Fungal alpha-amylases, predominantly derived from species like Aspergillus, excel in starch degradation for the food and beverage industry, facilitating dough conditioning, and enhancing fermentation processes. Plant-derived alpha-amylases, found in seeds and tubers, play pivotal roles in seed germination and starch metabolism. Beyond their natural functions, alpha-amylases find widespread use in diverse sectors, including baking, brewing, textile, and detergent industries, owing to their ability to hydrolyze starch into fermentable sugars and facilitate the production of various products. Thus, the diverse types of alpha-amylases offer an array of applications across multiple sectors, underscoring their significance in biotechnology and industrial processes.

[0003] Amylases can be distinguished based on the products they generate during starch hydrolysis. One notable type is maltogenic amylases, which exhibit specificity in producing maltose as a primaryproduct. Unlike typical alpha-amylases that generate a mixture of maltose, maltotriose, and other dextrins, maltogenic amylases preferentially produce maltose through the cleavage of a-l,4-glycosidic bonds within amylose, amylopectin and related glucose polymers, liberating maltose units from the non-reducing end of the polymer chain. This specificity makes maltogenic amylases particularly valuable in industries requiring high maltose yields, such as brewing and confectionery. Moreover, their ability to produce maltose efficiently can improve the flavor, texture, and shelf life of food products. Maltogenic amylases also find applications in the production of maltose syrups, where the enzymatic conversion of starch into maltose-rich syrups offers advantages in sweetness, solubility, and fermentability compared to traditional methods. In food industry, maltogenic amylase has been employed as a prominent baking enzyme aiming to improve softness, elasticity, freshness and to prolong shelf-life of baked products.

[0004] In the realm of wheat product baking, the inherent amylase content present in wheat, while naturally occurring, often fails to significantly enhance the quality of dough. Consequently, to augment dough characteristics and optimize the baking process, supplemental amylases sourced from fungi and bacteria are commonly incorporated. These exogenous enzymes, such as fungal alpha-amylases from species like Aspergillus, and bacterial alpha-amylases, particularly those from Bacillus strains, contribute to dough conditioning by efficiently breaking down starch molecules into simpler sugars. By catalyzing the hydrolysis of a-l,4-glycosidic bonds within starch, these amylases generate maltose and other dextrins, thereby facilitating improved fermentation, dough handling, and final product texture. The addition of fungal and bacterial amylases not only enhances dough machinability but also promotes desirable attributes in baked goods, including increased volume, enhanced crumb structure, and prolonged freshness. Moreover, these enzymes play a crucial role in achieving uniform browning and flavor development during baking, leading to superior sensory qualities in the finished wheat-based products. Thus, the supplementation of fungal and bacterial amylases in wheat baking exemplifies a strategic approach to optimizing dough performance and elevating the overall quality of baked goods.

[0005] Bacterial amylases are often preferred over fungal amylases for baking products due to several advantageous characteristics. Bacterial amylases generally possess a higher degree of specificity towards a-l,4-glycosidic bonds within starch molecules, resulting in more controlled hydrolysis and the predominant production of maltose. This specificity can lead to improved dough handling properties, enhanced fermentation, and superior crumb structure in the final baked goods. In contrast, fungal amylases may exhibit broader substrate specificity, potentially leading to a more heterogeneous mixture of sugar products and affecting the overall quality of the baked product. Additionally, bacterial amylases are often available in highly purified forms with consistent enzyme activity, making them easier to incorporate into standardized baking processes. Their commercial availability and cost -effectiveness further contribute to their preference in baking applications.

[0006] While amylases are efficient at hydrolyzing starch molecules into fermentable sugars, their activity can be inhibited by high concentrations of sugars present in the dough. This inhibition occurs due to competitive binding between the sugar molecules and the active site of the enzyme, reducing the enzyme's effectiveness in breaking down starch. Consequently, in dough formulations with elevated sugar levels, the enzymatic activity of amylases may be compromised, leading to incomplete starch hydrolysis and suboptimal fermentation. As a result, the dough may exhibit reduced volume, poor texture, and compromised flavor development during baking.

[0007] In the state of the art, approaches have been described for the development of alpha-amylase variants to enhance their performance and broaden their applicability. WO1999043794A1 discloses maltogenic alpha amylase variants from Bacillus strain NCIB 11837 with modified physicochemical properties, e.g. an altered pH optimum or improved thermostability. W02006032281A2 relates to a method of preparing a dough-based product with high sucrose content using sucrose-tolerant variants of maltogenic amylase, which maintains enzyme activity in high sucrose environments. WO2014131842A1 relates to an alpha-amylase variant from Alicyclobacillus pohliae NCIMB14276 strain with enhanced thermostability and sucrose tolerance.

[0008] However, there remains a need for new engineered alpha-amylases tailored specifically to address the persistent challenges encountered in baking and food processing industries. Despite the pivotal role of alpha-amylases in optimizing dough performance and enhancing product quality, current variants often fall short in terms of enzymatic activity and sucrose tolerance. This shortfall underscores the pressing necessity for innovative solutions to overcome these limitations and pave the way for more efficient and reliable enzymatic processes. By developing alpha-amylase variants with enhanced enzymatic activity and improved sucrose tolerance, researchers can propel the industry towards greater efficiency, consistency, and quality in dough conditioning and starch hydrolysis, ultimately meeting the evolving demands of manufacturers and consumers alike.SUMMARY OF THE INVENTION

[0009] The present disclosure presents a comprehensive invention concerning variant polypeptides with alpha-amylase activity. The variants of the present invention involve amino acid substitutions at specific positions compared to wild-type sequences, which lead to alterations in the functional properties of the alpha-amylase enzymes. Furthermore, the disclosure highlights the potential applications of these variant polypeptides in various industrial processes and compositions, owing to their improved catalytic efficiency compared to wild-type enzymes.

[0010] In one aspect of the invention, a variant polypeptide with alpha-amylase activity is disclosed. The variant polypeptide comprised an amino acid sequence having at least 85% identity to the amino acid wild-type sequence (SEQ ID NO: 1) and comprises an amino acid substitution in at least one amino acid position of SEQ ID NO: 1 selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and F639.

[0011] It has been surprisingly found that the variant polypeptide with alpha-amylase activity comprising an amino acid substitution, as described above, resulted in at least one altered and / or improved feature as compared with the wild-type SEQ ID NO: 1. Specifically, it has been surprisingly found that the variant polypeptides with alpha-amylase activity of the present invention have at least one of altered features (A) and (B), or a combination thereof; wherein:(A) an increased enzymatic activity, when compared to the wild-type alpha-amylase of SEQ ID NO: 1 under standard test conditions; and(B) an increased sucrose tolerance, when compared to the wild-type alpha-amylase of SEQ ID NO:1 under standard test conditions.

[0012] It has also been surprisingly found that the variant polypeptide with alpha-amylase activity comprising an amino acid substitution, as described above, resulted in at least one altered feature generally pertaining to the utilization of an alpha-amylase in the creation of doughs and baked products. Specifically, it has been surprisingly found that the variant polypeptides with alpha-amylase activity of the present invention leads to at least one altered feature to a dough or baked product in comparison with the wild-type alpha-amylase of SEQ ID NO: 1, such as (A) a reduced hardness after storage of the baked product; (B) a reduced loss of resilience over storage of the baked product; (C) an improved cohesiveness of the baked product; and / or (D) an improved water mobility of the baked product. Such improvements have also been observed in doughs or baked products comprising a certain amount of sucrose, including high amounts of sucrose.

[0013] In a second aspect of the invention, a method of expressing a variant polypeptide having alpha amylase activity according to the invention is provided. Such a method comprises (a) cultivating a recombinant host cell under conditions appropriated for the production of a variant polypeptide having alpha amylase activity according to the invention; and (b) recovering a variant polypeptide having alpha amylase activity according to the invention.

[0014] In a further aspect of the invention, a composition comprising a variant polypeptide having alpha-amylase activity according to the invention and one or more components is provided. Specifically, a dough conditioner is obtained by combining the variant polypeptide with alpha-amylase activity as described herein with a selection of additional components selected from the group comprising diluents, additional enzymes, emulsifiers, oxidizing agents, reducing agents, hydrocolloids, surfactants, dough conditioner agents, flavor enhancers, preservatives, and nutritional supplements, or a combination thereof. Furthermore, a pre-mix composition is introduced, comprising a flour or a mixture of flour along with the variant polypeptide of the present invention.

[0015] In another aspect of the invention, the use of a variant polypeptide having alpha-amylase activity according to the invention or of a composition according to the invention or of a pre -mix according to the invention in the preparation of a dough and / or a baked product is disclosed. This application extends to scenarios where the dough and / or baked product contains a high sucrose content. Furthermore, the high sucrose content can be characterized by a specific sucrose concentration.

[0016] In a further aspect of the invention a dough or a baked product comprising a variant polypeptide having alpha-amylase activity according to the invention or a composition according to the invention is disclosed.

[0017] In a final aspect of the invention, a method of preparing a dough or a baked product is disclosed. Such a method comprises adding a variant polypeptide having alpha-amylase activity according to the invention to the dough or baked product.DEFINITIONS

[0018] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well as the singular forms, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising”, when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0019] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0020] In describing the invention, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0021] For the purposes of the present disclosure, the term “wild-type polypeptide” refers to the naturally occurring form of a polypeptide, typically found in the organism's genome without any genetic modifications or alterations. Specifically, in the case of alpha-amylase activity, the wild-type polypeptide would be the naturally existing alpha-amylase enzyme encoded by the corresponding gene sequence in an organism's genome. This wild-type polypeptide (such as the wild-type alpha amylase of SEQ ID NO: 1) serves as a reference point for comparing variant polypeptides generated through genetic engineering or mutagenesis techniques, allowing for the assessment of alterations in structure and function.

[0022] For the purposes of the present disclosure, the term "alpha-amylase activity" means the ability of a polypeptide to catalyze the hydrolysis of alpha- 1,4-glycosidic linkages in starch molecules, resulting in the production of smaller oligosaccharides, such as maltose and glucose. This enzymatic activity is crucial for various industrial processes, including the production of food and beverages, detergents, and biofuels. The measurement of alpha-amylase activity can be conducted using standardized assays, such as the determination of reducing sugars released from starch substrates.

[0023] For the purposes of the present disclosure, the term "variant polypeptides" encompasses polypeptides with alterations in their amino acid sequence compared to a reference polypeptide, typically a wild-type polypeptide having alpha-amylase activity, such as the alpha-amylase of SEQ ID NO: 1. These alterations can include substitutions, deletions, or insertions of one or more amino acids, resulting in polypeptides with modified properties or functions.

[0024] For the purposes of the present disclosure, the variant polypeptides having alpha-amylase activity may also be referred to as "alpha-amylase," "amylase," or "maltogenic amylase" without alteringtheir intended meaning. These terms are used interchangeably to describe the enzyme's ability to catalyze the hydrolysis of starch molecules into smaller fermentable sugars, such as maltose and glucose.

[0025] SEQ ID NO: 1 is the amino acid sequence of the maltogenic amylase from Geobacillus stea-rothermophilus without its natural N-terminal Signal Peptide sequence. For the purposes of the disclosure, the amino acid sequence of SEQ ID NO:1 is presented using the three-letter code, while amino acid substitutions of SEQ ID NO: 1 are described using the one-letter code for amino acids. The three-letter code follows IUPAC nomenclature, with amino acids listed sequentially from the N-terminus to the C-terminus, separated by spaces. Every 5thposition is indicated by a position number below the sequence.SEQ ID NO:1Ser Ser Ser Ala Ser Vai Lys Gly Asp Vai lie Tyr Gin lie lie lie5 10 15Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn Asn Pro Ala Lys Ser 20 25 30Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp Gly 35 40 45Gly Asp Leu Glu Gly Vai Arg Gin Lys Leu Pro Tyr Leu Lys Gin Leu 50 55 60Gly Vai Thr Thr lie Trp Leu Ser Pro Vai Leu Asp Asn Leu Asp Thr 65 70 75 80 Leu Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp Thr Arg Asp 85 90 95Phe Lys Gin lie Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp Thr 100 105 110Leu Vai Asn Asp Ala His Gin Asn Gly lie Lys Vai lie Vai Asp Phe 115 120 125Vai Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe Ala130 135 140 Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr Phe 145 150 155 160 Asp Asp Ala Thr Lys Gly Tyr Phe His His Asn Gly Asp lie Ser Asn 165 170 175 Trp Asp Asp Arg Tyr Glu Ala Gin Trp Lys Asn Phe Thr Asp Pro Ala 180 185 190Gly Phe Ser Leu Ala Asp Leu Ser Gin Glu Asn Gly Thr lie Ala Gin 195 200 205Tyr Leu Thr Asp Ala Ala Vai Gin Leu Vai Ala His Gly Ala Asp Gly 210 215 220Leu Arg lie Asp Ala Vai Lys His Phe Asn Ser Gly Phe Ser Lys Ser 225 230 235 240 Leu Ala Asp Lys Leu Tyr Gin Lys Lys Asp lie Phe Leu Vai Gly Glu 245 250 255 Trp Tyr Gly Asp Asp Pro Gly Thr Ala Asn His Leu Glu Lys Vai Arg 260 265 270Tyr Ala Asn Asn Ser Gly Vai Asn Vai Leu Asp Phe Asp Leu Asn Thr 275 280 285Vai lie Arg Asn Vai Phe Gly Thr Phe Thr Gin Thr Met Tyr Asp Leu 290 295 300Asn Asn Met Vai Asn Gin Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn 305 310 315 320 Leu lie Thr Phe lie Asp Asn His Asp Met Ser Arg Phe Leu Ser Vai 325 330 335 Asn Ser Asn Lys Ala Asn Leu His Gin Ala Leu Ala Phe lie Leu Thr 340 345 350Ser Arg Gly Thr Pro Ser lie Tyr Tyr Gly Thr Glu Gin Tyr Met Ala 355 360 365Gly Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp Thr 370 375 380Thr Thr Thr Ala Phe Lys Glu Vai Ser Thr Leu Ala Gly Leu Arg Arg 385 390 395 400 Asn Asn Ala Ala lie Gin Tyr Gly Thr Thr Thr Gin Arg Trp lie Asn 405 410 415 Asn Asp Vai Tyr lie Tyr Glu Arg Lys Phe Phe Asn Asp Vai Vai Leu 420 425 430Vai Ala lie Asn Arg Asn Thr Gin Ser Ser Tyr Ser lie Ser Gly Leu 435 440 445Gin Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly Leu 450 455 460Leu Gly Gly Asn Gly lie Ser Vai Ser Asn Gly Ser Vai Ala Ser Phe 465 470 475 480 Thr Leu Ala Pro Gly Ala Vai Ser Vai Trp Gin Tyr Ser Thr Ser Ala 485 490 495 Ser Ala Pro Gin lie Gly Ser Vai Ala Pro Asn Met Gly lie Pro Gly 500 505 510Asn Vai Vai Thr lie Asp Gly Lys Gly Phe Gly Thr Thr Gin Gly Thr 515 520 525Vai Thr Phe Gly Gly Vai Thr Ala Thr Vai Lys Ser Trp Thr Ser Asn 530 535 540Arg lie Glu Vai Tyr Vai Pro Asn Met Ala Ala Gly Leu Thr Asp Vai 545 550 555 560 Lys Vai Thr Ala Gly Gly Vai Ser Ser Asn Leu Tyr Ser Tyr Asn lie565 570 575 Leu Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala Pro 580 585 590Pro Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro Glu 595 600 605Leu Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala Gln 610 615 620Gly Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe Ser 625 630 635 640 Vai Pro Ala Gly Lys Thr lie Gin Phe Lys Phe Phe lie Lys Arg Ala 645 650 655Asp Gly Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr 660 665 670Pro Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn675 680 685

[0026] It is within the meaning of the invention, that whenever an embodiment of any of the aspects of the invention refer to an amylase, any such embodiment is meant to also refer to an amylase providing the same embodiments and / or to a polypeptide providing the same embodiments. In this respect the invention also relates to an amylase, and / or to a polypeptide comprising an amino acid sequence with at least 85 % identity to SEQ ID NO:1, wherein the amino acid sequence is substituted compared to SEQ ID NO: 1 in at least one amino acid position selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and F639. Accordingly, all embodiment of any aspect referring to the nuclease of the invention shall also refer to an endonuclease and / or a polypeptide correspondingly.

[0027] The alpha-amylase according to the invention comprises an amino acid sequence with a defined identity to the amino acid sequence of SEQ ID NO: 1. This means that any variant polypeptide according to the invention may comprise said amino acid sequence as a subsequence of its overall amino acidsequence, or may essentially consist of said amino acid sequence. When the variant polypeptide / alpha-amylase according to the invention comprises said amino acid sequence as a subsequence of its overall amino acid sequence, said overall amino acid sequence may be extended, i.e. may comprise additional amino acid residues, at the N-terminus and / or at the C-terminus of said subsequence. Such extension may be advantageous, for example, when the enzyme is to be immobilized on a solid support, e.g. for purification purposes.

[0028] For the purposes of the present disclosure, "standard test conditions" refer to well-established methodologies and environmental parameters that are routinely recognized and utilized by those skilled in the art. These conditions typically include, but are not limited to, specific temperature ranges, humidity levels, pressure settings, and standardized testing protocols that ensure consistency and reliability of results. The techniques and conditions described herein are provided as illustrative examples. However, a skilled person in the field will appreciate how to modify and adapt these techniques and conditions based on specific requirements or varying circumstances.

[0029] For the purpose of the present disclosure, in preferred embodiments, the standard test conditions comprises assays for enzymatic activity (or alpha-amylase activity), wherein an assay for enzymatic activity (or alpha-amylase activity) of a polypeptide is defined as the activity for hydrolytic cleavage of a carbohydrate substrate into two carbohydrate products, preferably as the activity for the hydrolytic cleavage of the substrate maltotriose into the products glucose and maltose.

[0030] Routinely, the standard test conditions to determine enzymatic activity, in particular alpha-amylase activity assays of a polypeptide comprise the steps of (a) reacting defined amounts of test polypeptide having alpha-amylase activity (a-1) in a pre-heated buffered assay reaction mixture with a final buffer concentration in a range of 0.005 M to 0.25 M buffer compound, preferably with buffer compound NaOAc, and preferably with a final buffer concentration of 0.05 M NaOAc buffer (pH 5.0), (a-2) together with a suitable carbohydrate substrate in a suitable concentration range of 0.01 % (w / w) to 30 % (w / w), preferably with the substrate maltotriose at 1 % (w / w) or 2 % (w / w), (a-3) for a defined time between 1 minute to 240 minutes and temperature between 30 °C to 85 °C, preferably for 60 minutes at 40 °C, (b) subsequent stopping of the reaction, for example by addition of 1.25-fold excessof 2 M Tris-HCl buffer (pH 7.55), (c) measuring the concentration of the formed carbohydrate product formed by any suitable assay, preferably by measuring glucose resulting from hydrolysis of maltotriose using the D-glucose HK Assay Kit (Megazymes K-GLUHK-220A) according to the microplate assay procedure from the manual and measuring the absorbance at 340 nm, and (d) quantifying the amounts of carbohydrate product formed with a suitable assay, preferably quantifying glucose formed by use of a calibration curve taken from glucose standard solutions with concentrations within the relevant concentration ranges.

[0031] For the purpose of the present disclosure, in preferred embodiments, the standard test conditions to determine sucrose tolerance, is defined as the assay for alpha-amylase enzyme activity, wherein the assay reaction mixture described above is supplemented with sucrose at a final concentration in the range of 10 mM to 1000 mM sucrose, preferably at final concentration of 50 mM to 300 mM sucrose, more preferably at final concentration of 100 mM or 300 mM sucrose.

[0032] For the purposes of the present disclosure, the percent identity is calculated as: Sequence Identity [%] = number of Matches / L x 100, wherein L is the number of aligned positions, i.e. identities and non-identities (including gaps, if any). Identity is preferably calculated using BLASTP (see, for example, Altschul SF et al. (1997) " Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; or Altschul SF (2005) " Protein database searches using compositionally adjusted substitution matrices." FEBS J. 272:5101-5109); preferably with the following algorithm parameters: Matrix: BLOSUM62; Gap Costs: Existence: 11 Extension: 1, Expect threshold: 0.05, and Word size: 6. BlastP can be accessed online at the NCBI Homepage (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi? PROGRAM=blastp& PAGE TYPE=BlastSearch& LINK _LOC=blasthome). Other program settings can be adjusted as desired, for example using the following settings:- Field " Enter Query Sequence": Query subrange: none;- Field " Choose Search Set": Database: non-redundant protein sequences (nr); optional parameters: none- Field " Program Selection": Algorithm: blastp (protein-protein BLAST);- Algorithm parameters: Field " General parameters": Max target sequences: 5000; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 0.05; Word size: 6; Max matches in a query range: 0;- Algorithm parameters: Field " Scoring parameters": Matrix: BLOSUM62; Gap Costs: Existence: 11 Extension: 1; Compositional adjustments: Conditional compositional score matrix adjustment; -Algorithm parameters: Field " Filters and Masking": Filter: none; Mask: none.

[0033] In addition to the default parameters for calculation of percent identity, when aligning a reference sequence, referred to as "query sequence", with another sequence, referred to as "subject sequence", in particular wherein the query sequence is any of the sequences provided by the present invention; preferably SEQ ID NO: 1, and wherein the subject sequence is any other sequence, for example a sequence disclosed in a database, the subject sequence must align over a sequence stretch covering at least 85% of the overall length of the query sequence ("sequence coverage" of at least 85%); the alignment of both sequences must cover at least 85% of the query sequence; for the purpose of specification, the sequence coverage is calculated as: the number of aligned positions, i.e. covering identities and nonidentities (including gaps, if any) divided by the overall length of the query sequence and multiplied by 100. Alignments with a lower sequence coverage of the query sequence by the subject sequence are excluded for the determination of sequence identity for the purposes of this application. However, the subject sequence may be longer than the length of the alignment.

[0034] During expression of naturally or recombinantly expressed variant polypeptides in certain host organisms, alpha-amylase genes may be linked with a so-called signal peptide (“SP”) gene sequence. The SP gene sequence is co-expressed together with the target-enzyme to form a single precursor protein comprising the SP and the alpha-amylase protein part. The SP element of the SP-alpha-amylase fusion protein usually serves as a secretion signal and is cleaved from the nuclease during the secretion process. For the purpose of the present specification, gene sequences or peptide sequences of a signal peptide of alpha-amylases are disregarded in calculation of the identity of a nuclease according to theinvention. Tools for identifying SP gene or protein sequences are known to the person skilled in the art and are described for example under https: / / www.uniprot.org / help / signal, Section 2, in which predictive tools Phobius, Predotar, SignalP and TargetP are given as examples.

[0035] For the purposes of the present disclosure, a certain target amino acid sequence being substituted at a certain “amino acid position number compared to SEQ ID NO:1” means that such target amino acid sequence is substituted at such target amino acid position number of the target amino acid sequence that aligns with the “amino acid position number of SEQ ID NO: 1” in an alignment which is prepared in accordance with the requirements of sequence identity thereof, irrespective of whether the amino acid position number of SEQ ID NO: 1 and the target amino acid position number are identical or different.

[0036] For the purposes of the present disclosure, a certain target amino acid sequence being substituted at a certain “amino acid position number compared to SEQ ID NO: 1” furthermore means that in any certain amino acid position number of SEQ ID NO:1 the amino acid of SEQ ID NO:1 in such position is substituted by any differing amino acid selected from the group of natural proteinogenic amino acids, i.e. A, R, N, D, C, Q, E, G, H, I, L, K, M, F, T, W, Y, V, P or S.

[0037] For the purpose of the present specification, said at least amino acid position of the alpha-amylase being substituted means that said at least one amino acid position is substituted by (i) either a positively charged amino acid selected from R, K, and H, or (ii) a negatively charged amino acid selected from E and D, or (iii) an aliphatic non-polar amino acid selected from G, A, V, L, I, and P, or (iv) an aromatic amino acid selected from W, F, and Y, or (v) an aliphatic polar non-charged amino acid selected from N, Q, S, T, C, and M.

[0038] For the purpose of the present specification, said at least amino acid position of the alpha-amylase being substituted may be (i) a positively charged amino acid which is substituted by another positively charged amino acid, or (ii) a negatively charged amino acid which is substituted by another negatively charged amino acid, or (iii) an aliphatic non-polar amino acid which is substituted by another aliphatic non-polar amino acid, or (iv) which is an aromatic amino acid which is substituted by anotheraromatic amino acid, or (v) an aliphatic polar non-charged amino acid which is substituted by another aliphatic polar non-charged amino acid.

[0039] For the purpose of the present specification, said at least amino acid position of the alpha-amylase being substituted may be (i) a positively charged amino acid and is substituted by an aliphatic non-polar amino acid, or an aromatic amino acid, or an aliphatic polar non-charged, or a negatively charged amino acid; or (ii) a negatively charged amino acid and is substituted by an aliphatic non-polar amino acid, or an aromatic amino acid, or an aliphatic polar non-charged, or a positively charged amino acid; or (iii) an aliphatic non-polar amino acid and is substituted by an aromatic amino acid, or an aliphatic polar non-charged, or a positively charged amino acid, or a negatively charged amino acid; or (iv) an aromatic amino acid and is substituted by an aliphatic non-polar amino acid, or an aliphatic polar non-charged, or a positively charged amino acid, or a negatively charged amino acid; or (v) an aliphatic polar non-charged amino acid and is substituted by an aliphatic non-polar amino acid, or an aromatic amino acid, or a positively charged amino acid, or a negatively charged amino acid.

[0040] For the purposes of the present disclosure, the status of charge of any amino acids being substituted in any position compared to SEQ ID NO: 1, in particular the charge ofthe positively charged amino acids R, K, and H and the charge of the negatively charged amino acids E and D is determined at a pH of 7.

[0041] For the purposes of the present disclosure, specific substitution positions disclosed herein are described in accordance with common practice in the field of amino acid substitutions, wherein the format [non-substituted amino acid - sequence position] is chosen and the sequence position is directly flanked with the amino acids letter (one letter code) of the not yet mutated amino acid on the left side, wherein no specific amino acid is selected as a substituent for such amino acid position. For clarification only, substitution " P51” describes the substitution of the amino acid proline (P) in sequence position 51 by any substituent.

[0042] For the purposes of the present disclosure, specific substitutions disclosed herein are described in accordance with common practice in the field of amino acid substitutions, wherein the format [non-substituted amino acid - sequence position - substituted amino acid] is chosen and the sequence position is directly flanked with the amino acids letter (one letter code) on the left or right side. For clarification only, substitution " P51R” describes the substitution of the amino acid proline (P) in sequence position 51 by the amino acid arginine (R), i.e. P is replaced by R.

[0043] For the purposes of the present disclosure, the term "nucleic acid" refers to a molecule that comprises a sequence of nucleotides, such as DNA or RNA, capable of encoding genetic information or serving as a substrate for biochemical reactions. Nucleic acids of the present invention can be generated using standard molecular biology techniques well known to those skilled in the art, taken in combination with the sequence information provided herein.

[0044] For example, nucleic acid molecules may be synthesized de novo using standard synthetic techniques. Such synthesis is typically an automated process. Additionally, nucleic acid molecules can be generated through site-directed mutagenesis of existing nucleic acid molecules, such as wild-type nucleic acid molecules. Site-directed mutagenesis can be carried out using various techniques known in the art, including PCR-based methods. In this method, PCR is performed on a plasmid template using oligonucleotide primers encoding the desired substitution. The resulting mutated fragments can be selectively amplified and preserved while eliminating the template DNA.

[0045] Moreover, nucleic acids of the invention can be generated using cDNA, mRNA, or genomic DNA as a template and appropriate mismatched oligonucleotide primers according to the site -directed mutagenesis technique. These nucleic acid molecules can be cloned into suitable vectors and characterized by DNA sequence analysis. Additionally, nucleic acid sequences of the invention may comprise deletions, which can be introduced using site-directed mutagenesis techniques. Oligonucleotides corresponding to or hybridizable to nucleotide sequences according to the invention can be prepared using standard synthetic techniques, including automated DNA synthesis. Complementary nucleic acid molecules, sufficiently complementary to other nucleotide sequences to form stable duplexes, are also included in the present invention.

[0046] For the purposes of the present disclosure, the term "nucleic acid construct" refers to a specifically engineered nucleic acid molecule designed for a particular purpose, which may include gene expression, gene editing, or other genetic manipulation techniques.

[0047] Nucleic acid constructs of the present invention can be adapted using standard molecular biology techniques well known to those skilled in the art. For instance, a nucleic acid construct may be synthesized de novo using standard synthetic techniques. This typically involves automated synthesis processes. Alternatively, a nucleic acid construct can be generated through site-directed mutagenesis of an existing nucleic acid molecule, such as a wild-type nucleic acid molecule. Site-directed mutagenesis can be carried out using various techniques, including PCR-based methods, where specific substitutions or deletions are introduced into the nucleic acid sequence.

[0048] Nucleic acid constructs can be designed to include regulatory elements, such as promoters, enhancers, and terminators, to control gene expression. Additionally, these constructs may incorporate reporter genes or selection markers for downstream analysis or manipulation. Characterization of nucleic acid constructs often involves DNA sequence analysis to confirm the integrity and fidelity of the engineered sequence. Moreover, nucleic acid constructs can be cloned into suitable vectors for expression in recombinant host cells, facilitating various applications in biotechnology and molecular biology.

[0049] For the purposes of the present disclosure, the term "recombinant expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked, designed for the expression of a specific gene in a host cell. Recombinant expression vectors typically comprise regulatory sequences, such as promoters, enhancers, and terminators, selected based on the host cells intended for expression. These vectors can autonomously replicate in a host cell or integrate into the host genome upon introduction. Within a recombinant expression vector, the gene of interest is operatively linked to the regulatory sequences, enabling expression of the gene in the host cell.

[0050] The design of recombinant expression vectors often involves the selection of suitable regulatory elements to control gene expression. These elements include constitutive or inducible promoters, enhancers, and other expression control elements, such as polyadenylation signals. Regulatory sequencescan direct expression in a variety of host cells or tissues, including bacterial, yeast, insect, mammalian cells, or even in vitro transcription / translation systems.

[0051] Recombinant expression vectors may take the form of plasmids, viral vectors, or other genetic elements capable of directing gene expression. Commonly used vectors include plasmids derived from bacterial sources or viral vectors such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses. Additionally, expression vectors can contain selectable markers, allowing for the identification and selection of host cells that have successfully incorporated the vector.

[0052] The introduction of recombinant expression vectors into host cells can be achieved through various methods, including transformation, transfection, transduction, infection, or electroporation. Once introduced into the host cell, the expression vector directs the transcription and translation of the gene of interest, resulting in the production of the desired protein or peptide. The expressed proteins can then be harvested and characterized using standard molecular biology techniques.

[0053] For the purposes of the present disclosure, the term "recombinant host cell" refers to a cell into which (or into an ancestor of which) a nucleic acid according to the invention has been introduced using recombinant DNA techniques. These techniques involve the insertion of the nucleic acid into the host cell's genome or into an autonomously replicating extrachromosomal element, such as a plasmid. Both prokaryotic and eukaryotic cells can serve as recombinant host cells, including bacteria, fungi, yeast, and mammalian cell lines.

[0054] Examples of suitable bacterial host organisms include various gram-positive bacterial species, such as Bacillus subtilis, Bacillus licheniformis, Bacillus cereus. and Streptomyces species, as well as gram-negative species like Escherichia coli and Pseudomonas species. These bacterial hosts are often preferred fortheir ease of manipulation and rapid growth rates.

[0055] For eukaryotic host organisms, yeast cells are commonly used, such as Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces lactis. Additionally, mammalian cell lines like CHO (Chinese hamster ovary), HEK293 (human embryonic kidney), and COS (CV-1 origin of SV40) cells are frequently employed for the expression of complex proteins due to their ability to perform proper post-translational modifications. Filamentous fungi, including species of Aspergillus, Penicillium, and Trichoderma, are also utilized as recombinant host cells. These fungi offer advantages such as high protein secretion capacity and compatibility with industrial-scale fermentation processes.

[0056] Recombinant host cells are chosen based on factors such as their ability to modulate gene expression, post-translational modifications, and processing of the encoded protein. This ensures the production of functional and properly modified proteins. The selection of a suitable host cell depends on various factors, including the desired protein characteristics, scale of production, and downstream applications.

[0057] Stably transfected cell lines, capable of producing the desired protein variant over multiple generations, can also serve as recombinant host cells. Methods for constructing stable cell lines, including vector design and transfection techniques, are well-known in the art and can be tailored to specific host systems.

[0058] For the purposes of the present disclosure, the term “maltogenic alpha-amylase activity” refers to the ability of an enzyme to catalyze the hydrolysis of starch molecules, specifically targeting the alpha- 1,4-glycosidic bonds present within the starch structure. Such enzymes are also referred to as 1,4-alpha-D-glucan glucanohydrolase, belonging to the enzyme classification of EC 3.2.1.1 This activity results in the production of glucose, maltose, dextrin and other oligosaccharides from starch substrates.

[0059] For the purposes of the present disclosure, the term “starch” refers to a polymer primarily composed of two types of glucose polymers, namely amylose and amylopectin. Amylose is a linear chain of glucose molecules linked by alpha- 1,4-glycosidic bonds, while amylopectin is a branched polymer with alpha- 1,4-glycosidic bonds forming the main chain and alpha- 1,6-glycosidic bonds at branch points. Examples of starch sources include, but are not limited to, flours, such as wheat flour, rice flour, com flour (maize flour), barley flour, oat flour, potato flour, cassava flour, tapioca flour, chickpea flour, almond flour, coconut flour, sorghum flour, and mixtures thereof.

[0060] For the purposes of the present disclosure, the term "flour" refers to finely ground grains or starch-rich ingredients derived from various sources, primarily wheat, but may also include other cereals such as rice, corn, or barley or mixtures thereof. Specifically, "flour" in this context emphasizes the starch content of the ingredient, as it serves as the primary substrate for enzymatic action by alpha-amylase. This starch-rich component of flour provides the necessary substrate for the enzymatic hydrolysis catalyzed by alpha-amylase, leading to the production of fermentable sugars such as maltose and glucose during dough fermentation or baking processes.

[0061] For the purposes of the present disclosure, the term “related glucose polymers” refers to any polymer comprising glucose molecules linked by alpha- 1,4-glycosidic bonds other than starch in which an enzyme having alpha-amylase activity can act, such as glycogen, dextrins, and other products obtained from the partial hydrolysis thereof. Examples of suitable dextrins include, but are not limited to white dextrins, yellow dextrins, British gum, pregelatinized dextrins, and maltodextrins.

[0062] For the purposes of the present disclosure, the variant polypeptide having an alpha-amylase activity according to the invention can act on different types of starches. Different types of starch may include, but are not limited to, soluble starch, resistant starch, and damaged starch.

[0063] For the purposes of the present disclosure, the term “Soluble Starch” refers to the type of starch that readily dissolves in water to form a viscous solution. Maltogenic alpha-amylase can act on soluble starch, breaking down its alpha- 1,4-glycosidic bonds to produce maltose and other oligosaccharides.

[0064] For the purposes of the present disclosure, the term “Resistant Starch” refers to the type of starch that resists digestion in the small intestine. It can be physically inaccessible starch, such as that found in whole or partially milled grains and seeds, which is encapsulated by fibrous cell walls and resistant to enzymatic action; resistant granular starch, characterized by its crystalline and compact granular structure, found mostly in raw and green vegetables, such as potatoes, bananas, and legumes; retrograded starch, formed when gelatinized starch (e.g., cooked and then cooled) undergoes retrogradation, resulting in the reformation of crystalline structures with reduced susceptibility to digestion; and chemically modified starch, which is synthesized or chemically modified to resist enzymatic digestion. Maltogenic alpha-amylase may act on certain forms of resistant starch, depending on their structure andaccessibility to enzymatic hydrolysis. This can lead to the production of maltose and other oligosaccharides.

[0065] For the purposes of the present disclosure, the term “Damaged Starch” refers to starch granules that have undergone physical or mechanical damage, such as during milling or processing. Maltogenic alpha-amylase can act on damaged starch, hydrolyzing its alpha- 1,4-glycosidic bonds and potentially improving its functionality in various food applications.

[0066] For the purposes of the present disclosure, maltose is the main product resulting from the hydrolysis of starch and related glucose polymers by maltogenic alpha-amylase. Maltose is a disaccharide consisting of two glucose units linked by an alpha- 1,4-glycosidic bond.

[0067] In addition to maltose, maltogenic alpha-amylase activity can lead to the formation of various oligosaccharides from starch hydrolysis. These oligosaccharides may include:Maltotriose: consisting of three glucose units linked by alpha- 1,4-glycosidic bonds;Maltotetraose: consisting of four glucose units linked by alpha- 1,4-glycosidic bonds;Maltopentaose: consisting of five glucose units linked by alpha- 1,4-glycosidic bonds; and Higher oligosaccharides: comprising longer chains of glucose units, depending on the degree of hydrolysis and the specificity of the enzyme.The specific types and proportions of oligosaccharides produced can vary depending on factors such as the substrate (e.g., type of starch), reaction conditions (e.g., pH, temperature), and the characteristics of the maltogenic alpha-amylase enzyme itself.

[0068] The variant polypeptide having alpha-amylase activity according to the invention exhibits at least one altered feature in comparison with SEQ ID NO: 1, wherein the altered feature may be increased sucrose tolerance, increased enzymatic activity, increased stability / activity at different pH range, increased activity on raw starch, and altered substrate specificity or a combination thereof.

[0069] For the purposes of the present disclosure, "increased enzymatic activity" indicates that the variant polypeptide exhibits a higher catalytic efficiency or turnover rate or enzymatic activity compared to the wild-type enzyme under specific conditions, preferably under standard test conditions for enzymatic activity. This enhancement in enzymatic activity results in a greater rate of substrate conversion or product formation, which can lead to improved process efficiency and productivity in various applications.

[0070] For the purposes of the present disclosure, the enzymatic activity of a variant polypeptide according to the invention refers to the conversion of any of the substrates described herein, especially starch or any substrate rich in starch, into maltose. The alpha-amylase activity is detected by any alpha-amylase activity assay known in the art. Standard tests and conditions for measuring enzymatic activity typically involve the use of specific substrates, controlled pH levels, temperature settings, and detection methods to ensure accurate and reproducible results. Such standard conditions are well understood by those skilled in the art and are routinely used in enzymatic assays. For example, the assay may use maltotriose as the substrate; the maltogenic amylase hydrolyzes the substrate to yield glucose and maltose; and finally the produced glucose is detected using the D-glucose HK Assay Kit (Megazymes K-GLUHK-220A) or any other suitable glucose assay kit.

[0071] For the purposes of the present disclosure, "increased sucrose tolerance" refers to the enhanced ability of the variant polypeptide to maintain its enzymatic activity in the presence of higher concentrations of sucrose compared to the wild-type enzyme. This alteration allows the enzyme to function effectively in environments with elevated sucrose levels, which may be beneficial in industrial processes where sucrose is present as a substrate or co-solute. Sucrose tolerance may be expressed as enzymatic activity in the presence of increasing concentrations of sucrose, wherein the enzymatic activity may be determined using any suitable alpha-amylase activity assay known in the art, preferably by using the sucrose standard test conditions for sucrose tolerance. Standard tests and conditions for assessing enzymatic activity typically involve varying substrate concentrations, controlled pH levels, temperature settings, and detection methods to ensure accurate and reproducible results. These standard conditions are well understood by those skilled in the art and are routinely used in enzymatic assays.

[0072] The variant polypeptide having alpha-amylase activity according to the invention exhibits at least one altered feature in comparison with SEQ ID NO: 1, wherein the altered feature may be increased stability / activity at different pH range, increased activity on raw starch, and altered substrate specificity or a combination thereof.

[0073] For the purposes of the present disclosure, "increased stability / activity at different pH range" denotes the broader pH tolerance range exhibited by the variant polypeptide compared to the wild-type enzyme. This alteration allows the enzyme to maintain its enzymatic activity over a wider pH spectrum, enhancing its versatility and applicability in diverse industrial processes where pH conditions may vary.

[0074] For the purposes of the present disclosure, "increased activity on raw starch" signifies the enhanced ability of the variant polypeptide to hydrolyze raw or uncooked starch compared to the wildtype enzyme. This alteration allows the enzyme to efficiently degrade starch substrates without the need for extensive preprocessing, which can simplify and streamline industrial processes involving starch conversion.

[0075] For the purposes of the present disclosure, "altered substrate specificity" indicates changes in the preference or efficiency of the variant polypeptide towards specific substrates compared to the wildtype enzyme. This alteration may result in expanded substrate utilization or improved selectivity for particular substrates, offering opportunities for tailored enzyme applications in various industries.

[0076] For the purposes of the present disclosure, the terms “dough conditioner”, “flour treatment agent”, “improving agent” and “bread improver” refer to a substance, ingredient or composition used in the preparation of dough to improve its handling properties, texture, and final product quality. Dough conditioners can encompass a variety of compounds, including enzymes, emulsifiers, oxidants, reducing agents, hydrocolloids, and other functional ingredients. These additives help to modify the rheological properties of the dough, making it easier to knead, shape, and handle during processing. Additionally, dough conditioners may enhance the dough's ability to retain moisture, improve its elasticity and strength, and contribute to the development of desirable texture, crumb structure, and crust characteristics in the finished baked goods. The term "dough conditioner" as used herein encompasses the variantpolypeptides having alpha-amylase of the present invention and a broad range of ingredients aimed at optimizing the performance and quality of dough in various baking applications.

[0077] For the purposes of the present disclosure, the term "pre-mix" refers to a blended mixture of ingredients or components that are combined together in advance of use in a specific application or process, particularly in the context of dough preparation for baked goods. Pre-mixes typically contain a combination of dry ingredients such as flour, sugar, salt, leavening agents, and other functional additives, including but not limited to enzymes, emulsifiers, stabilizers, and flavorings. These pre-mixed formulations offer convenience and consistency in baking operations by providing a standardized blend of ingredients ready for further processing. By pre-mixing key components, manufacturers can streamline production processes, reduce preparation time, and ensure uniformity in the final product. The term "pre-mix" as used herein encompasses a composition comprising a source of flour and the variant polypeptides having alpha-amylase activity of the present invention, or the dough conditioner as described above, and is tailored to specific baking applications, including bread, pastry, cake, and other baked goods.

[0078] For the purposes of the present disclosure, the term "baked product" refers to any food item that has been prepared through the process of baking, which typically involves cooking in an oven using heat. Baked products encompass a wide variety of goods, including but not limited to bread, pastries, cakes, cookies, muffins, pies, and crackers. These products are often characterized by their distinct texture, flavor, aroma, and appearance, which are imparted through the chemical and physical transformations that occur during the baking process. Baked products may vary widely in their ingredients, formulations, shapes, sizes, and cultural significance, catering to diverse culinary preferences and dietary needs.

[0079] Examples of baked products include, but are not limited to bread of various types such as white, brown, or whole-meal bread (including white, whole-meal, or rye bread), typically available as loaves or rolls, French baguette -style bread, pastries like croissants, brioche, and panettone, pasta, noodles prepared by boiling or stir-frying, pita bread, and other flatbreads, tortillas, tacos, cakes, pancakes, cookies (especially biscuits), doughnuts (including yeasted doughnuts), bagels, pie crusts, steamedbread, crispbread, brownies, sheet cakes, snack foods like pretzels, tortilla chips, and fabricated snacks, as well as fabricated potato crisps. Additionally, baked products include a wide range of items such as tin bread, twisted bread, buns (such as hamburger buns or steamed buns), chapati, rusk, dried steam bun slices, bread crumbs, matzos, focaccia, melba toast, zwieback, croutons, soft pretzels, both soft and hard bread, breadsticks, various types of leavened bread (both yeast and chemically -leavened), laminated dough products like Danish pastry, croissants, or puff pastry products, muffins, Danish pastries, bagels, confectionery coatings, crackers, wafers, pizza crusts, tortillas, pasta products, crepes, waffles, parbaked products, as well as refrigerated and frozen dough products.

[0080] For the purposes of the present disclosure, the term "dough" refers to a mixture of ingredients, typically including flour, water, yeast, salt, and optionally a source of sugar that is kneaded together to form a cohesive mass. This mixture serves as the base for various baked goods such as bread, pastries, and pizza crusts. Dough may also contain other ingredients such as fat, eggs, and flavorings, depending on the specific recipe and desired characteristics of the final product. Throughout the baking process, the dough undergoes fermentation, during which yeast converts sugars into carbon dioxide gas, causing the dough to rise and develop a light and airy texture. The term "dough" as used herein encompasses both raw dough prior to baking and partially or fully baked dough products. The term dough herein also includes a batter. A batter is a semi-liquid mixture, being thin enough to drop or pour from a spoon, of one or more flours combined with liquids such as water, milk or eggs used to prepare various foods, including cake.

[0081] For the purposes of the present disclosure, a sugar-containing baked product and dough refers to baked goods and dough formulations that incorporate sugar as a key ingredient. These products and doughs typically include a significant amount of sugar in their composition, contributing to sweetness, flavor, texture, and browning during the baking process. The term "sugar-containing" emphasizes the presence of sugar as a defining component of these baked products and doughs, which may include items such as bread, sweet bread, cakes, pastries, cookies, and other confectionery items. Typically, a baked product or dough encompasses sugar levels ranging from 2 to 30 wt%.

[0082] For the purposes of the present disclosure, the term "sugar" refers to a class of carbohydrates that are sweet-tasting, soluble in water, and commonly used as sweeteners in food and beverage production. Sugars can be categorized into various types, including sucrose (table sugar), fructose (found in fruits and honey), glucose (found in plants and used as a primary energy source), lactose (found in milk), and maltose (formed during the breakdown of starch). In the context of baked products and doughs, sugar serves multiple functions, including providing sweetness, contributing to texture and mouthfeel, promoting browning through caramelization, and acting as a preservative by inhibiting microbial growth. The term "sugar" encompasses both natural sugars found in ingredients like fruits and dairy products, as well as added sugars used in food processing and culinary applications. Sucrose, a disaccharide composed of glucose and fructose molecules, stands out as the primary sugar ingredient chosen for its desirable properties in baked goods and doughs. Its neutral flavor, solubility, and ability to enhance sweetness, texture, and browning during baking make sucrose the preferred choice for formulating high-quality products. While other sugars may be utilized, sucrose is favored for its consistent performance and widespread availability, ensuring the production of baked goods with superior taste, appearance, and shelf life.

[0083] Novel variant polypeptides having alpha-amylase activity and uses thereof are described herein. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be evident, however, to one skilled in the art that the present invention may be practiced without these specific details.

[0084] The present disclosure is to be considered as an exemplification of the invention and is not intended to limit the invention to the specific embodiments illustrated by description below.DETAILED DESCRIPTION OF THE INVENTIONFirst aspect: variant polypeptides having alpha-amylase activity:

[0085] The first aspect of the present invention encompasses variant polypeptides having alpha-amylase activity as compared with a reference polypeptide having alpha-amylase activity. The referencepolypeptide may typically be a wild-type polypeptide having alpha-amylase activity. The reference polypeptide may also be referred to as a parent polypeptide or comparison polypeptide.

[0086] In one embodiment, the wild-type reference polypeptide may be obtained from any suitable organisms.

[0087] Geobacillus is a genus of thermophilic bacteria known for their ability to thrive in high -temperature environments. Alpha-amylases from Geobacillus species are notable for their robust enzymatic activity and stability under extreme conditions, including high temperatures and alkaline pH. These enzymes play pivotal roles in various industrial applications, including starch processing, biofuel production, and detergent formulations.

[0088] In one preferred embodiment, the wild-type reference polypeptides may be obtained from a species pertaining to the genus Geobacillus selected from the group comprising, for example, Geobacillus stearothermophilus, Geobacillus thermoleovorans, Geobacillus thermoglucosidasius, Geobacillus caldoxylosilyticus, Geobacillus kaustophilus. Preferably, the wild-type reference polypeptides of the present invention are obtained from Geobacillus stearothermophilus. Preferably, the reference polypeptide obtained from Geobacillus stearothermophilus has the amino acid sequence set out in SEQ ID NO: 1.

[0089] In one embodiment, a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 is provided.

[0090] In preferred embodiments, the variant polypeptide according having alpha-amylase activity of the present invention comprises an amino acid sequence having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.

[0091] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1.

[0092] In preferred embodiments, the variant polypeptide having alpha-amylase activity of the present invention has at least 85 % identity to SEQ ID NO:1 and comprises an amino acid substitution in at least one amino acid position selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and F639.

[0093] In preferred embodiments, the variant polypeptide having alpha-amylase activity of the present invention comprises an amino acid substitution in at least one amino acid position compared to SEQ ID NO:1, wherein the amino acid position is selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and F639.

[0094] In preferred embodiments, the amino acid position is selected from the group consisting of K270, L571, V585, E101, M156, and F639. Preferably, the amino acid position is selected from the group consisting of K270, L571, V585, and F639. Even more preferably, the amino acid position is K270.

[0095] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position K270, wherein the substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, K270A, K270Q, K270E, K270M, K270P, K270T, K270W, K270Y; preferably the substitution is selected from K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F or K270S; even more preferably the substitution is selected from K270R and K270H. In a most preferred embodiment of the invention; the variant polypeptide with alpha-amylase activity according to the invention has the amino acid substitution K270R.

[0096] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position T25, wherein the substitution is selected from the group consisting of T25A, T25R, T25N, T25D, T25C, T25Q, T25E, T25G, T25H, T25I, T25L, T25K, T25M, T25F, T25P, T25S, T25W, T25Y, and T25V; preferably the substitution is selected from the group consisting of T25A, T25G, T25I, T25L, T25P and T25V, even more preferably the substitution is T25A.

[0097] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position Q99, wherein the substitution is selected from the group consisting of Q99R, Q99A, Q99N, Q99D, Q99C, Q99E, Q99G, Q99H, Q99I, Q99L, Q99K, Q99M, Q99F, Q99P, Q99S, Q99T, Q99W, Q99Y, and Q99V; preferably the substitution is Q99R, Q99K or Q99H, even more preferably the substitution is Q99R.

[0098] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position E101, wherein the substitution is selected from the group consisting of E101D, E101A, E101R, E101N, E101C, E101Q, E101G, E101H, E101I, E101L, E101K, E101M, E101F, E101P, E101S, E101T, E101W, E101Y, and E101V; preferably the substitution is selected between E101D and E101E.

[0099] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position A138, wherein the substitution is selected from the group consisting of A138V, A138R, A138N, A138D, A138C, A138Q, A138E, A138G, A138H, A138I, A138L, A138K, A138M, A138F, A138P, A138S, A 138T, A 138W, and A 138Y; preferably the substitution is selected from the group consisting of A 138G, A 138V, A 138L, A 1381, and A 138P; even more preferably the substitution is A 138V.

[0100] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position Y155, wherein the substitution is selected from the group consisting ofY155L, Y155A, Y155R, Y155N, Y155D, Y155C, Y155Q, Y155E, Y155G, Y155H, Y155I, Y155K, Y155M, Y155F, Y155P, Y155S, Y155T, Y155W, and Y 155V; preferably the substitution is selected from the group consisting ofY155G, Y155A, Y155V, Y155L, Y155I, andY155P; even more preferably the substitution isY155L.

[0101] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position M156, wherein the substitution is selected from the group consisting of M156I, M156A, M156R, M156N, M156D, M156C, M156Q, M156E, M156G, M156H, M156L, M156K, M156F, M156P,M156S, M156T, M156W, M156Y, and M156V; preferably the substitution is selected from the group consisting of M156G, M156A, M156V, M156L, and M156P; even more preferably the substitution is M156I.

[0102] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position W185, wherein the substitution is selected from the group consisting of W185Y, W185A, W185R, W185N, W185D, W185C, W185Q, W185E, W185G, W185H, W185I, W185L, W185K, W185M, W185F, W185P, W185S, W185T, and W185V; preferably the substitution is selected between W185Y and W185F.

[0103] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position A265, wherein the substitution is selected from the group consisting of A265P, A265S, A265R, A265N, A265D, A265C, A265Q, A265E, A265G, A265H, A265I, A265L, A265K, A265M, A265F, A265T, A265W, A265Y, and A265V; preferably the substitution is selected from the group consisting of A265G, A265V, A265L, A265I, A265N, A265Q, A265T, A265C, and A265M; even more preferably the substitution is selected between A265P and A265S.

[0104] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position N309, wherein the substitution is selected from the group consisting of N309T, N309A, N309R, N309D, N309C, N309Q, N309E, N309G, N309H, N309I, N309L, N309K, N309M, N309F, N309P, N309S, N309W, N309Y, and N309V; preferably the substitution is selected from the group consisting of N309Q, N309S, N309T, N309C, and N309M; even more preferably the substitution is N309T.

[0105] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position K318, wherein the substitution is selected from the group consisting of K318E, K318I, K318A, K318R, K318N, K318D, K318C, K318Q, K318G, K318H, K318L, K318M, K318F, K318P, K318S, K318T, K318W, K318Y, and K318 V; preferably the substitution is selected from the group consisting of K318E,K318D, K318I, K138G, K138A, K138V, K138L, and K138P; even more preferably the substitution is selected between K318E and K318I.

[0106] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position A368, wherein the substitution is selected from the group consisting of A368S, A368R, A368N, A368D, A368C, A368Q, A368E, A368G, A368H, A368I, A368L, A368K, A368M, A368F, A368P, A368T, A368W, A368Y, and A368V; preferably the substitution is selected from the group consisting of A368N, A368Q, A368T, A368C, A368M and A368S; even more preferably the substitution is A368S.

[0107] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position A478, wherein the substitution is selected from the group consisting of A478S, A478R, A478N, A478D, A478C, A478Q, A478E, A478G, A478H, A478I, A478L, A478K, A478M, A478F, A478P, A478T, A478W, A478Y, and A478V; preferably the substitution is selected from the group consisting of A478N, A478Q, A478T, A478C, A478M and A478S; even more preferably the substitution is A478S.

[0108] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position L571, wherein the substitution is selected from the group consisting of L571G, L571S, L571A, L571R, L571N, L571D, L571C, L571Q, L571E, L571H, L571I, L571K, L571M, L571F, L571P, L571T, L571W, L571Y, and L571 V; preferably the substitution is selected from the group consisting of L571G, L571A, L571V, L571I, L571P, L571N, L571Q, L571S, L571T, L571C and L571M; even more preferably the substitution is selected between L571G and L571S.

[0109] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position 1576, wherein the substitution is selected from the group consisting of I576L, I576V, I576A, I576R, I576N, I576D, I576C, I576Q, I576E, I576G, I576H, I576K, I576M, I576F, I576P, I576S, I576T, I576W, and I576Y; preferably the substitution is selected from the group consisting of I576G, I576A, I576P I576L or I576V; even more preferably the substitution is selected between I576L and I576V.

[0110] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position V585, wherein the substitution is selected from the group consisting of V585R, V585A, V585N, V585D, V585C, V585Q, V585E, V585G, V585H, V585I, V585L, V585K, V585M, V585F, V585P, V585S, V585T, V585W, and V585Y; preferably the substitution is selected from the group consisting of V585D, V585R, V585K, and V585H; even more preferably the substitution is selected between V585Rand V585D.

[0111] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position V638, wherein the substitution is selected from the group consisting of V638D, V638A, V638R, V638N, V638C, V638Q, V638E, V638G, V638H, V638I, V638L, V638K, V638M, V638F, V638P, V638S, V638T, V638W, and V638Y; preferably the substitution is selected between V638D and V638E.

[0112] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 at least in position F639, wherein the substitution is selected from the group consisting of F639V, F639A, F639R, F639N, F639D, F639C, F639Q, F639E, F639G, F639H, F639I, F639L, F639K, F639M, F639P, F639S, F639T, F639W, and F639Y; preferably the substitution is selected from the group consisting of F639V, F639G, F639A, F639L, F639I, F639P, even more preferably the substitution is F639V.

[0113] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, said at least one amino acid position is substituted by:a positively charged amino acid selected from R, K, and H; ora negatively charged amino acid selected from E and D;an aliphatic non-polar amino acid selected from G, A, V, L, I, and P; oran aromatic amino acid selected from W, F, and Y; oran aliphatic polar non-charged amino acid selected from N, Q, S, T, C, and M.

[0114] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, said at least one amino acid position is:a positively charged amino acid and substituted by another positively charged amino acid; or a negatively charged amino acid and substituted by another negatively charged amino acid; or an aliphatic non-polar amino acid and substituted by another aliphatic non-polar amino acid; or an aromatic amino acid and substituted by another aromatic amino acid; oran aliphatic polar non-charged amino acid and substituted by another aliphatic polar noncharged amino acid.

[0115] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, said at least one amino acid position is:a positively charged amino acid and is substituted by an aliphatic non-polar amino acid, or an aromatic amino acid, or an aliphatic polar non-charged, or a negatively charged amino acid; or a negatively charged amino acid and is substituted by an aliphatic non-polar amino acid, or an aromatic amino acid, or an aliphatic polar non-charged, or a positively charged amino acid; or an aliphatic non-polar amino acid and is substituted by an aromatic amino acid, or an aliphatic polar non-charged, or a positively charged amino acid, or a negatively charged amino acid; or an aromatic amino acid and is substituted by an aliphatic non-polar amino acid, or an aliphatic polar non-charged, or a positively charged amino acid, or a negatively charged amino acid; or an aliphatic polar non-charged amino acid and is substituted by an aliphatic non-polar amino acid, or an aromatic amino acid, or a positively charged amino acid, or a negatively charged amino acid.

[0116] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least one amino acid position when compared to SEQ ID NO: 1, wherein said at least one amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A,Q99R, E101D, A138V, Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, A478S, L571G, L571S, I576L, I576V, V585R, V638D, and F639V.

[0117] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, said at least one amino acid substitution is selected from the group consisting of K270R, E101D, M156I, L571G, L571S, V585R, and F639V. Preferably, said at least one amino acid substitution is selected from the group consisting of K270R, E101D, M156I, L571G, L571S, V585D, and F639V. More preferably, said at least one amino acid substitution is selected from the group consisting of K270R, L571S and V585R. More preferably, said at least one amino acid substitution is selected from the group consisting of K270R, L571S and V585D. Even more preferably, said at least one amino acid substitution is K270R.

[0118] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention is characterized by the amino acid sequence SEQ ID NO:2, which comprises the amino acid substitution K270R, or a variant thereof.

[0119] In preferred embodiments, the variant polypeptide with alpha-amylase activity according to the invention may advantageously have an amino acid substitution in more than one position compared to SEQ ID NO: 1.

[0120] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid positions when compared to SEQ ID NO:1.

[0121] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, said at least two amino acid positions may be selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and F639. In most preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, said at least two amino acid positions may be selected from the group consisting of K270, E101, M156, L571, V585, and F639. Preferably, said at least two amino acid positions are selected from the group consisting of K270, L571, V585, and F639. In preferred embodiments,the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid positions when compared to SEQ ID NO:1, wherein the amino acid positions may be selected from the pair group consisting of K270+Y273, K270+D285, K270+N309, K270+K318, K270+M367, K270+A368, K270+F427, K270+Q449, K270+A478, K270+S488, K270+S493, K270+T494, K270+T528, K270+V534, K270+M553, K270+L571, K270+I576, K270+V585, K270+A629, K270+V638, K270+F639, T25+T68, T25+D85, T25+R95, T25+Q99, T25+E101, T25+I125, T25+P130, T25+F136, T25+A138, T25+S141, T25+T142, T25+N151, T25+Y155, T25+M156, T25+A163, T25+R180, T25+E182, T25+W185, T25+D198, T25+L199, T25+Q201, T25+H232, T25+F233, T25+S235, T25+Y258, T25+G259, T25+D260, T25+A265, T25+K270, T25+Y273, T25+D285, T25+N309, T25+K318, T25+M367, T25+A368, T25+F427, T25+Q449, T25+A478, T25+S488, T25+S493, T25+T494, T25+T528, T25+V534, T25+M553, T25+L571, T25+I576, T25+V585, T25+A629, T25+V638, T25+F639, Q99+E101, Q99+I125, Q99+P130, Q99+F136, Q99+A138, Q99+S141, Q99+T142, Q99+N151, Q99+Y155, Q99+M156, Q99+A163, Q99+R180, Q99+E182, Q99+W185, Q99+D198, Q99+L199, Q99+Q201, Q99+H232, Q99+F233, Q99+S235, Q99+Y258, Q99+G259, Q99+D260, Q99+A265, Q99+K270, Q99+Y273, Q99+D285, Q99+N309, Q99+K318, Q99+M367, Q99+A368, Q99+F427, Q99+Q449, Q99+A478, Q99+S488, Q99+S493, Q99+T494, Q99+T528, Q99+V534, Q99+M553, Q99+L571, Q99+I576, Q99+V585, Q99+A629, Q99+V638, Q99+F639, E101+I125, E101+P130, E101+F136, E101+A138, E101+S141, E101+T142, E101+N151, E101+Y155, E101+M156, E101+A163, E101+R180, E101+E182, E101+W185, E101+D198, E101+L199, E101+Q201, E101+H232, E101+F233, E101+S235, E101+Y258, E101+G259, E101+D260, E101+A265, E101+K270, E101+Y273, E101+D285, E101+N309, E101+K318, E101+M367, E101+A368, E101+F427, E101+Q449, E101+A478, E101+S488, E101+S493, E101+T494, E101+T528, E101+V534, E101+M553, E101+L571, E101+I576, E101+V585, E101+A629, E101+V638, E101+F639, A138+S141, A138+T142, A138+N151, A138+Y155, A138+M156, A138+A163, A138+R180, A138+E182, A138+W185, A138+D198, A138+L199, A138+Q201, A138+H232, A138+F233, A138+S235, A138+Y258, A138+G259, A138+D260, A138+A265, A138+K270, A138+Y273, A138+D285, A138+N309, A138+K318, A138+M367, A138+A368, A138+F427, A138+Q449,A138+A478, A138+S488, A138+S493, A138+T494, A138+T528, A138+V534, A138+M553, A138+L571, A138+I576, A138+V585, A138+A629, A138+V638, A138+F639, Y155+M156, Y155+A163, Y155+R180, Y155+E182, Y155+W185, Y155+D198, Y155+L199, Y155+Q201, Y155+H232, Y155+F233, Y155+S235, Y155+Y258, Y155+G259, Y155+D260, Y155+A265, Y155+K270, Y155+Y273, Y155+D285, Y155+N309, Y155+K318, Y155+M367, Y155+A368, Y155+F427, Y155+Q449, Y155+A478, Y155+S488, Y155+S493, Y155+T494, Y155+T528, Y155+V534, Y155+M553, Y155+L571, Y155+I576, Y155+V585, Y155+A629, Y155+V638, Y155+F639, M156+A163, M156+R180, M156+E182, M156+W185, M156+D198, M156+L199, M156+Q201, M156+H232, M156+F233, M156+S235, M156+Y258, M156+G259, M156+D260, M156+A265, M156+K270, M156+Y273, M156+D285, M156+N309, M156+K318, M156+M367, M156+A368, M156+F427, M156+Q449, M156+A478, M156+S488, M156+S493, M156+T494, M156+T528, M156+V534, M156+M553, M156+L571, M156+I576, M156+V585, M156+A629, M156+V638, M156+F639, W185+D198, W185+L199, W185+Q201, W185+H232, W185+F233, W185+S235, W185+Y258, W185+G259, W185+D260, W185+A265, W185+K270, W185+Y273, W185+D285, W185+N309, W185+K318, W185+M367, W185+A368. W185+F427, W185+Q449, W185+A478, W185+S488, W185+S493, W185+T494, W185+T528, W185+V534, W185+M553, W185+L571, W185+I576, W185+V585, W185+A629, W185+V638, W185+F639, A265+K270, A265+Y273, A265+D285, A265+N309, A265+K318, A265+M367, A265+A368, A265+F427, A265+Q449, A265+A478, A265+S488, A265+S493, A265+T494, A265+T528, A265+V534, A265+M553, A265+L571, A265+I576, A265+V585, A265+A629, A265+V638, A265+F639, N309+K318, N309+M367, N309+A368, N309+F427, N309+Q449, N309+A478, N309+S488, N309+S493, N309+T494, N309+T528, N309+V534, N309+M553, N309+L571, N309+I576, N309+V585, N309+A629, N309+V638, N309+F639, K318+M367, K318+A368, K318+F427, K318+Q449, K318+A478, K318+S488, K318+S493, K318+T494, K318+T528, K318+V534, K318+M553, K318+L571, K318+I576, K318+V585, K318+A629, K318+V638, K318+F639, A368+F427, A368+Q449, A368+A478, A368+S488, A368+S493, A368+T494, A368+T528, A368+V534, A368+M553, A368+L571, A368+I576, A368+V585, A368+A629, A368+V638, A368+F639, A478+S488, A478+S493, A478+T494, A478+T528, A478+V534, A478+M553,A478+L571, A478+I576, A478+V585, A478+A629, A478+V638, A478+F639, L571+I576, L571+V585, L571+A629, L571+V638, L571+F639, I576+V585, I576+A629, I576+V638, I576+F639, V585+A629, V585+V638, V585+F639 and V638+F639.

[0122] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid positions when compared to SEQ ID NO:1, wherein the amino acid positions are selected from the group consisting of K270+Y273, K270+M367, K270+M553, K270+L571, K270+V585, K270+F639, E101+M156, E101+R180, E101+F233, E101+Q449, E101+D260, E101+K270, E101+Y273, E101+M367, E101+M553, E101+L571, E101+V585, E101+F639, M156+R180, M156+F233, M156+Q449, M156+D260, M156+K270, M156+Y273, M156+M367, M156+M553, M156+L571, M156+V585, M156+F639, L571+V585, L571+F639, and V585+F639.

[0123] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid positions when compared to SEQ ID NO: 1, wherein said at least two amino acid positions are selected from the group consisting of K270+L571, K270+V585, K270+D260, K270+R180, K270+M553, K270+Y273, L571+V585. Preferably, said at least two amino acid positions are selected from the group consisting of K270+L571, K270+V585 and K270+R180.

[0124] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid sequence comprising at least two amino acid substitutions compared to SEQ ID NO: 1, wherein:when the amino acid position K270 is substituted, the amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, K270A, K270Q, K270E, K270M, K270P, K270T, K270W, K270Y; preferably the substitution is selected from K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F and K270S, more preferably the substitution is K270R;when the amino acid position E 101 is substituted, the amino acid substitution is selected from the group consisting of E101D, E101A, E101R, E101N, E101C, E101Q, E101G, E101H,E101I, E101L, E101K, E101M, E101F, E101P, E101S, E101T, E101W, E101Y, and E101V, preferably the substitution is E101D;when the amino acid position M156 is substituted, the amino acid substitution is selected from the group consisting of M156I, M156A, M156R, M156N, M156D, M156C, M156Q, M156E, M156G, M156H, M156L, M156K, M156F, M156P, M156S, M156T, M156W, M156Y, and M156V, preferably the substitution is M156I;when the amino acid position L571 is substituted, the amino acid substitution is selected from the group consisting of L571S, L571G, L571A, L571R, L571N, L571D, L571C, L571Q, L571E, L571H, L571I, L571K, L571M, L571F, L571P, L571T, L571W, L571Y, and L571V, preferably the substitution is selected between L571S and L571G; andwhen the amino acid position V585 is substituted, the amino acid substitution is selected from the group consisting of V585R, V585A, V585N, V585D, V585C, V585Q, V585E, V585G, V585H, V585I, V585L, V585K, V585M, V585F, V585P, V585S, V585T, V585W, and V585Y, preferably the substitution is selected between V585R and V585D; andwhen the amino acid position F639 is substituted, the amino acid substitution is selected from the group consisting of F639V, F639A, F639R, F639N, F639D, F639C, F639Q, F639E, F639G, F639H, F639I, F639L, F639K, F639M, F639P, F639S, F639T, F639W, and F639Y, preferably the substitution is F639V.

[0125] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid positions compared to SEQ ID NO: 1, wherein said at least two amino acid substitutions are selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A, Q99R, E101D, A138V, Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, A478S, L571G, L571S, I576L, I576V, V585D, V585R, V638D, and F639V. Preferably, said at least two amino acid positions are selected from the group consisting of K270R, E101D, M156I, R180G, F233M, D260G, D260S, M367L, Q449N, M553A, L571S, V585R, and F639V. More preferably, said at least two amino acid positions are selected from the group consisting of K270R, E101D, M156I,L571G, L571S, V585R, and F639V. More preferably, said at least two amino acid positions are selected from the group consisting of K270R, E101D, M156I, L571G, L571S, V585D, and F639V. More preferably, said at least two amino acid substitution positions are selected from the group consisting of K270R, L571S, Y273F, and V585R. More preferably, said at least two amino acid substitution positions are selected from the group consisting of K270R, L571S, Y273F, and V585D. Even more preferably, said at least two amino acid positions are selected from the group consisting of K270R, L571S and V585R. Even more preferably, said at least two amino acid positions are selected from the group consisting of K270R, L571S and V585D. Specifically, said at least two amino acid substitutions are K270R and L571S, K270R and V585R, or K270R, L571S and V585R. Specifically, said at least two amino acid substitutions are K270R and L571S, K270R and V585D, or K270R, L571S and V585D.

[0126] In preferred embodiments, when the variant has more than one substitution, such substitutions occur in at least one of the positions mentioned in this specification, in combination with any other amino acid position compared to SEQ ID NO: 1.

[0127] Such combinations include, but are not limited to, substitutions at more than one of the amino acid positions cited in this specification, substitutions where at least one of the amino acid positions cited in this specification is selected, or substitutions where any amino acid position compared to SEQ ID NO: 1 is selected.

[0128] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid position compared to SEQ ID NO: 1, wherein said at least two amino acid substitutions are selected from the group consisting of K270R+Y273F, K270R+M367L, K270R+Q449N, K270R+M553A, K270R+L571S, K270R+V585R, K270R+V585D, K270R+F639V, M156I+R180G, M156I+F233M, M156I+D260G, M156I+D260S, M156I+K270R, M156I+Y273F, M156I+M367L, M156I+Q449N, M156I+M553A, M156I+L571S, M156I+V585R, M156I+V585D, M156I+F639V, L571S+V585R, L571S+V585D, L571S+F639V, V585R+F639V, and V585R+F639V. Even more preferably the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid position compared to SEQ ID NO:1, wherein said at least two amino acid substitutions are selected from the group consisting of K270R+L571S, K270R+V585R, K270R+V585D, K270R+D260S,K270R+R180G, K270R+M553A and K270R+Y273F. Preferably, said at least two amino acid substitutions are selected from the group consisting of K270R+L571S, K270R+V585R, K270R+V585D and K270R+R180G. Also preferably, said at least two amino acid substitutions is K270R+L571S or K270R+V585R or K270R+V585D.

[0129] As above disclosed according to a preferred embodiment of the present invention, the variant polypeptide with alpha amylase activity comprises the amino acid substitution K270R, and is characterized by the amino acid sequence SEQ ID NO:2, or a variant thereof comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2.

[0130] In a preferred embodiment said variant polypeptide with alpha amylase activity further comprises one or more additional amino acid substitution in at least one amino acid position of SEQ ID NO:2, wherein the amino acid position is selected from the group consisting of T25, Q99, E101, A138, Y155, M156 W185, A265, A265, N309, K318, K318, A368, A478, L571, L571, 1576, 1576, V585, V638 and F639. More preferably said one or more additional amino acid substitution occurs in the amino acid position L571 and / or V585.

[0131] In a preferred embodiment said variant polypeptide with alpha amylase activity may further comprise one or more additional amino acid substitution in SEQ ID NO:2 selected from the group consisting of the specific substitutions occurring in the positions above listed in paragraphs

[0095] -

[0111] ,

[0132] In most preferred embodiments of the variant polypeptide with alpha amylase activity according to the invention, said one or more additional amino acid substitutions in SEQ ID NO:2 are selected from the group consisting of T25A, Q99R, E101D, A138V, Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, A478S, L571G, L571S, I576L, I576V, V585R, V585D, V638D and F639V. In a further preferred embodiment said one or more additional amino acid substitution is selected among the group consisting of L571G, L571S, V585D and V585R. According to a more preferredembodiment said one or more additional amino acid substitution is selected among the group consisting of L571G, L571S, V585D and V585R.

[0133] In preferred embodiments, the variant polypeptide with alpha-amylase activity according to the invention may have an amino acid substitution in more than one position compared to SEQ ID NO:2.

[0134] In preferred embodiments, when the variant has more than one substitution, such substitutions occur in at least one of the positions mentioned in this specification, in combination with any other amino acid position compared to SEQ ID NO:2.

[0135] Such combinations include, but are not limited to, substitutions at more than one of the amino acid positions cited in this specification, substitutions where at least one of the amino acid positions cited in this specification is selected, or substitutions where any amino acid position compared to SEQ ID NO:2 is selected.

[0136] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid positions compared to SEQ ID NO:2.

[0137] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention said amino acid substitutions in at least two amino acid positions are selected from the pair consisting of T25+T68, T25+D85, T25+R95, T25+Q99, T25+E101, T25+I125, T25+P130, T25+F136, T25+A138, T25+S141, T25+T142, T25+N151, T25+Y155, T25+M156, T25+A163, T25+R180, T25+E182, T25+W185, T25+D198, T25+L199, T25+Q201, T25+H232, T25+F233, T25+S235, T25+Y258, T25+G259, T25+D260, T25+A265, T25+K270, T25+Y273, T25+D285, T25+N309, T25+K318, T25+M367, T25+A368, T25+F427, T25+Q449, T25+A478, T25+S488, T25+S493, T25+T494, T25+T528, T25+V534, T25+M553, T25+L571, T25+I576, T25+V585, T25+A629, T25+V638, T25+F639, T68+D85, T68+R95, T68+Q99, T68+E101, T68+I125, T68+P130, T68+F136, T68+A138, T68+S141, T68+T142, T68+N151, T68+Y155, T68+M156, T68+A163, T68+R180, T68+E182, T68+W185, T68+D198, T68+L199, T68+Q201, T68+H232, T68+F233, T68+S235, T68+Y258, T68+G259, T68+D260, T68+A265, T68+K270, T68+Y273, T68+D285,T68+N309, T68+K318, T68+M367, T68+A368, T68+F427, T68+Q449, T68+A478, T68+S488, T68+S493, T68+T494, T68+T528, T68+V534, T68+M553, T68+L571, T68+I576, T68+V585, T68+A629, T68+V638, T68+F639, D85+R95, D85+Q99, D85+E101, D85+I125, D85+P130, D85+F136, D85+A138, D85+S141, D85+T142, D85+N151, D85+Y155, D85+M156, D85+A163, D85+R180, D85+E182, D85+W185, D85+D198, D85+L199, D85+Q201, D85+H232, D85+F233, D85+S235, D85+Y258, D85+G259, D85+D260, D85+A265, D85+K270, D85+Y273, D85+D285, D85+N309, D85+K318, D85+M367, D85+A368, D85+F427, D85+Q449, D85+A478, D85+S488, D85+S493, D85+T494, D85+T528, D85+V534, D85+M553, D85+L571, D85+I576, D85+V585, D85+A629, D85+V638, D85+F639, R95+Q99, R95+E101, R95+I125, R95+P130, R95+F136, R95+A138, R95+S141, R95+T142, R95+N151, R95+Y155, R95+M156, R95+A163, R95+R180, R95+E182, R95+W185, R95+D198, R95+L199, R95+Q201, R95+H232, R95+F233, R95+S235, R95+Y258, R95+G259, R95+D260, R95+A265, R95+K270, R95+Y273, R95+D285, R95+N309, R95+K318, R95+M367, R95+A368, R95+F427, R95+Q449, R95+A478, R95+S488, R95+S493, R95+T528, R95+V534, R95+M553, R95+L571, R95+I576, R95+V585, R95+A629, R95+V638, R95+F639, Q99+E101, Q99+I125, Q99+P130, Q99+F136, Q99+A138, Q99+S141, Q99+T142, Q99+N151, Q99+Y155, Q99+M156, Q99+A163, Q99+R180, Q99+E182, Q99+W185, Q99+D198, Q99+L199, Q99+Q201, Q99+H232, Q99+F233, Q99+S235, Q99+Y258, Q99+G259, Q99+D260, Q99+A265, Q99+K270, Q99+Y273, Q99+D285, Q99+N309, Q99+K318, Q99+M367, Q99+A368, Q99+F427, Q99+Q449, Q99+A478, Q99+S488, Q99+S493, Q99+T494, Q99+T528, Q99+V534, Q99+M553, Q99+L571, Q99+I576, Q99+V585, Q99+A629, Q99+V638, Q99+F639, E101+I125, E101+P130, E101+F136, E101+A138, E101+S141, E101+T142, E101+N151, E101+Y155, E101+M156, E101+A163, E101+R180, E101+E182, E101+W185, E101+D198, E101+L199, E101+Q201, E101+H232, E101+F233, E101+S235, E101+Y258, E101+G259, E101+D260, E101+A265, E101+K270, E101+Y273, E101+D285, E101+N309, E101+K318, E101+M367, E101+A368, E101+F427, E101+Q449, E101+A478, E101+S488, E101+S493, E101+T494, E101+T528, E101+V534, E101+M553, E101+L571, E101+I576, E101+V585, E101+A629, E101+V638, E101+F639, I125+P130, I125+F136, I125+A138, I125+S141, I125+T142, I125+N151, I125+Y155, I125+M156, I125+A163, I125+R180, I125+E182, I125+W185, I125+D198, I125+L199,I125+Q201, I125+H232, I125+F233, I125+S235, I125+Y258, I125+G259, I125+D260, I125+A265, I125+K270, I125+Y273, I125+D285, I125+N309, I125+K318, I125+M367, I125+A368, I125+F427, I125+Q449, I125+A478, I125+S488, I125+S493, I125+T494, I125+T528, I125+V534, I125+M553, I125+L571, I125+I576, I125+V585, I125+A629, I125+V638, I125+F639, P130+F136, P130+A138, P130+S141, P130+T142, P130+N151, P130+Y155, P130+M156, P130+A163, P130+R180, P130+E182, P130+W185, P130+D198, P130+L199, P130+Q201, P130+H232, P130+F233, P130+S235, P130+Y258, P130+G259, P130+D260, P130+A265, P130+K270, P130+Y273, P130+D285, P130+N309, P130+K318, P130+M367, P130+A368, P130+F427, P130+Q449, P130+A478, P130+S488, P130+S493, P130+T494, P130+T528, P130+V534, P130+M553, P130+L571, P130+I576, P130+V585, P130+A629, P130+V638, P130+F639, F136+A138, F136+S141, F136+T142, F136+N151, F136+Y155, F136+M156, F136+A163, F136+R180, F136+E182, F136+W185, F136+D198, F136+L199, F136+Q201, F136+H232, F136+F233, F136+S235, F136+Y258, F136+G259, F136+D260, F136+A265, F136+K270, F136+Y273, F136+D285, F136+N309, F136+K318, F136+M367, F136+A368, F136+F427, F136+Q449, F136+A478, F136+S488, F136+S493, F136+T494, F136+T528, F136+V534, F136+M553, F136+L571, F136+I576, F136+V585, F136+A629, F136+V638, F136+F639, A138+S141, A138+T142, A138+N151, A138+Y155, A138+M156, A138+A163, A138+R180, A138+E182, A138+W185, A138+D198, A138+L199, A138+Q201, A138+H232, A138+F233, A138+S235, A138+Y258, A138+G259, A138+D260, A138+A265, A138+K270, A138+Y273, A138+D285, A138+N309, A138+K318, A138+M367, A138+A368, A138+F427, A138+Q449, A138+A478, A138+S488, A138+S493, A138+T494, A138+T528, A138+V534, A138+M553, A138+L571, A138+I576, A138+V585, A138+A629, A138+V638, A138+F639, S141+T142, S141+N151, S141+Y155, S141+M156, S141+A163, S141+R180, S141+E182, S141+W185, S141+D198, S141+L199, S141+Q201, S141+H232, S141+F233, S141+S235, S141+Y258, S141+G259, S141+D260, S141+A265, S141+K270, S141+Y273, S141+D285, S141+N309, S141+K318, S141+M367, S141+A368, S141+F427, S141+Q449, S141+A478, S141+S488, S141+S493, S141+T494, S141+T528, S141+V534, S141+M553, S141+L571, S141+I576, S141+V585, S141+A629, S141+V638, S141+F639, T142+N151, T142+Y155, T142+M156, T142+A163,T142+R180, T142+E182, T142+W185, T142+D198, T142+L199, T142+Q201, T142+H232, T142+F233, T142+S235, T142+Y258, T142+G259, T142+D260, T142+A265, T142+K270, T142+Y273, T142+D285, T142+N309, T142+K318, T142+M367, T142+A368, T142+F427, T142+Q449, T142+A478, T142+S488, T142+S493, T142+T494, T142+T528, T142+V534, T142+M553, T142+L571, T142+I576, T142+V585, T142+A629, T142+V638, T142+F639, N151+Y155, N151+M156, N151+A163, N151+R180, N151+E182, N151+W185, N151+D198, N151+L199, N151+Q201, N151+H232, N151+F233, N151+S235, N151+Y258, N151+G259, N151+D260, N151+A265, N151+K270, N151+Y273, N151+D285, N151+N309, N151+K318, N151+M367, N151+A368, N151+F427, N151+Q449, N151+A478, N151+S488, N151+S493, N151+T494, N151+T528, N151+V534, N151+M553, N151+L571, N151+I576, N151+V585, N151+A629, N151+V638, N151+F639, Y155+M156, Y155+A163, Y155+R180, Y155+E182, Y155+W185, Y155+D198, Y155+L199, Y155+Q201, Y155+H232, Y155+F233, Y155+S235, Y155+Y258, Y155+G259, Y155+D260, Y155+A265, Y155+K270, Y155+Y273, Y155+D285, Y155+N309, Y155+K318, Y155+M367, Y155+A368, Y155+F427, Y155+Q449, Y155+A478, Y155+S488, Y155+S493, Y155+T494, Y155+T528, Y155+V534, Y155+M553, Y155+L571, Y155+I576, Y155+V585, Y155+A629, Y155+V638, Y155+F639, M156+A163, M156+R180, M156+E182, M156+W185, M156+D198, M156+L199, M156+Q201, M156+H232, M156+F233, M156+S235, M156+Y258, M156+G259, M156+D260, M156+A265, M156+K270, M156+Y273, M156+D285, M156+N309, M156+K318, M156+M367, M156+A368, M156+F427, M156+Q449, M156+A478, M156+S488, M156+S493, M156+T494, M156+T528, M156+V534, M156+M553, M156+L571, M156+I576, M156+V585, M156+A629, M156+V638, M156+F639, A163+R180, A163+E182, A163+W185, A163+D198, A163+L199, A163+Q201, A163+H232, A163+F233, A163+S235, A163+Y258, A163+G259, A163+D260, A163+A265, A163+K270, A163+Y273, A163+D285, A163+N309, A163+K318, A163+M367, A163+A368, A163+F427, A163+Q449, A163+A478, A163+S488, A163+S493, A163+T494, A163+T528, A163+V534, A163+M553, A163+L571, A163+I576, A163+V585, A163+A629, A163+V638, A163+F639, R180+E182, R180+W185, R180+D198, R180+L199, R180+Q201, R180+H232, R180+F233, R180+S235, R180+Y258, R180+G259, R180+D260, R180+A265, R180+K270, R180+Y273, R180+D285,R180+N309, R180+K318, R180+M367, R180+A368, R180+F427, R180+Q449, R180+A478, R180+S488, R180+S493, R180+T494, R180+T528, R180+V534, R180+M553, R180+L571, R180+I576, R180+V585, R180+A629, R180+V638, R180+F639, E182+W185, E182+D198, E182+L199, E182+Q201, E182+H232, E182+F233, E182+S235, E182+Y258, E182+G259, E182+D260, E182+A265, E182+K270, E182+Y273, E182+D285, E182+N309, E182+K318, E182+M367, E182+A368, E182+F427, E182+Q449, E182+A478, E182+S488, E182+S493, E182+T494, E182+T528, E182+V534, E182+M553, E182+L571, E182+I576, E182+V585, E182+A629, E182+V638, E182+F639, W185+D198, W185+L199, W185+Q201, W185+H232, W185+F233, W185+S235, W185+Y258, W185+G259, W185+D260, W185+A265, W185+K270, W185+Y273, W185+D285, W185+N309, W185+K318, W185+M367, W185+A368, W185+F427, W185+Q449, W185+A478, W185+S488, W185+S493, W185+T494, W185+T528, W185+V534, W185+M553, W185+L571, W185+I576, W185+V585, W185+A629, W185+V638, W185+F639, D198+L199, D198+Q201, D198+H232, D198+F233, D198+S235, D198+Y258, D198+G259, D198+D260, D198+A265, D198+K270, D198+Y273, D198+D285, D198+N309, D198+K318, D198+M367, D198+A368, D198+F427, D198+Q449, D198+A478, D198+S488, D198+S493, D198+T494, D198+T528, D198+V534, D198+M553, D198+L571, D198+I576, D198+V585, D198+A629, D198+V638, D198+F639, L199+Q201, L199+H232, L199+F233, L199+S235, L199+Y258, L199+G259, L199+D260, L199+A265, L199+K270, L199+Y273, L199+D285, L199+N309, L199+K318, L199+M367, L199+A368, L199+F427, L199+Q449, L199+A478, L199+S488, L199+S493, L199+T494, L199+T528, L199+V534, L199+M553, L199+L571, L199+I576, L199+V585, L199+A629, L199+V638, L199+F639, Q201+H232, Q201+F233, Q201+S235, Q201+Y258, Q201+G259, Q201+D260, Q201+A265, Q201+K270, Q201+Y273, Q201+D285, Q201+N309, Q201+K318, Q201+M367, Q201+A368, Q201+F427, Q201+Q449, Q201+A478, Q201+S488, Q201+S493, Q201+T494, Q201+T528, Q201+V534, Q201+M553, Q201+L571, Q201+I576, Q201+V585, Q201+A629, Q201+V638, Q201+F639, H232+F233, H232+S235, H232+Y258, H232+G259, H232+D260, H232+A265, H232+K270, H232+Y273, H232+D285, H232+N309, H232+K318, H232+M367, H232+A368, H232+F427, H232+Q449, H232+A478, H232+S488, H232+S493, H232+T494, H232+T528, H232+V534, H232+M553,H232+L571, H232+I576, H232+V585, H232+A629, H232+V638, H232+F639, F233+S235, F233+Y258, F233+G259, F233+D260, F233+A265, F233+K270, F233+Y273, F233+D285, F233+N309, F233+K318, F233+M367, F233+A368, F233+F427, F233+Q449, F233+A478, F233+S488, F233+S493, F233+T494, F233+T528, F233+V534, F233+M553, F233+L571, F233+I576, F233+V585, F233+A629, F233+V638, F233+F639, S235+Y258, S235+G259, S235+D260, S235+A265, S235+K270, S235+Y273, S235+D285, S235+N309, S235+K318, S235+M367, S235+A368, S235+F427, S235+Q449, S235+A478, S235+S488, S235+S493, S235+T494, S235+T528, S235+V534, S235+M553, S235+L571, S235+I576, S235+V585, S235+A629, S235+V638, S235+F639, Y258+G259, Y258+D260, Y258+A265, Y258+K270, Y258+Y273, Y258+D285, Y258+N309, Y258+K318, Y258+M367, Y258+A368, Y258+F427, Y258+Q449, Y258+A478, Y258+S488, Y258+S493, Y258+T494, Y258+T528, Y258+V534, Y258+M553, Y258+L571, Y258+I576, Y258+V585, Y258+A629, Y258+V638, Y258+F639, G259+D260, G259+A265, G259+K270, G259+Y273, G259+D285, G259+N309, G259+K318, G259+M367, G259+A368, G259+F427, G259+Q449, G259+A478, G259+S488, G259+S493, G259+T494, G259+T528, G259+V534, G259+M553, G259+L571, G259+I576, G259+V585, G259+A629, G259+V638, G259+F639, D260+A265, D260+K270, D260+Y273, D260+D285, D260+N309, D260+K318, D260+M367, D260+A368, D260+F427, D260+Q449, D260+A478, D260+S488, D260+S493, D260+T494, D260+T528, D260+V534, D260+M553, D260+L571, D260+I576, D260+V585, D260+A629, D260+V638, D260+F639, A265+K270, A265+Y273, A265+D285, A265+N309, A265+K318, A265+M367, A265+A368, A265+F427, A265+Q449, A265+A478, A265+S488, A265+S493, A265+T494, A265+T528, A265+V534, A265+M553, A265+L571, A265+I576, A265+V585, A265+A629, A265+V638, A265+F639, K270+Y273, K270+D285, K270+N309, K270+K318, K270+M367, K270+A368, K270+F427, K270+Q449, K270+A478, K270+S488, K270+S493, K270+T494, K270+T528, K270+V534, K270+M553, K270+L571, K270+I576, K270+V585, K270+A629, K270+V638, K270+F639, Y273+D285, Y273+N309, Y273+K318, Y273+M367, Y273+A368, Y273+F427, Y273+Q449, Y273+A478, Y273+S488, Y273+S493, Y273+T494, Y273+T528, Y273+V534, Y273+M553, Y273+L571, Y273+I576, Y273+V585, Y273+A629, Y273+V638, Y273+F639, D285+N309, D285+K318,D285+M367, D285+A368, D285+F427, D285+Q449, D285+A478, D285+S488, D285+S493, D285+T494, D285+T528, D285+V534, D285+M553, D285+L571, D285+I576, D285+V585, D285+A629, D285+V638, D285+F639, N309+K318, N309+M367, N309+A368, N309+F427, N309+Q449, N309+A478, N309+S488, N309+S493, N309+T494, N309+T528, N309+V534, N309+M553, N309+L571, N309+I576, N309+V585, N309+A629, N309+V638, N309+F639, K318+M367, K318+A368, K318+F427, K318+Q449, K318+A478, K318+S488, K318+S493, K318+T494, K318+T528, K318+V534, K318+M553, K318+L571, K318+I576, K318+V585, K318+A629, K318+V638, K318+F639, M367+A368, M367+F427, M367+Q449, M367+A478, M367+S488, M367+S493, M367+T494, M367+T528, M367+V534, M367+M553, M367+L571, M367+I576, M367+V585, M367+A629, M367+V638. M367+F639. A368+F427, A368+Q449, A368+A478, A368+S488, A368+S493, A368+T494, A368+T528, A368+V534, A368+M553, A368+L571, A368+I576, A368+V585, A368+A629, A368+V638, A368+F639, F427+Q449, F427+A478, F427+S488, F427+S493, F427+T494, F427+T528, F427+V534, F427+M553, F427+L571, F427+I576, F427+V585, F427+A629, F427+V638, F427+F639, Q449+A478, Q449+S488, Q449+S493, Q449+T494, Q449+T528, Q449+V534, Q449+M553, Q449+L571, Q449+I576, Q449+V585, Q449+A629, Q449+V638, Q449+F639, A478+S488, A478+S493, A478+T494, A478+T528, A478+V534, A478+M553, A478+L571, A478+I576, A478+V585, A478+A629, A478+V638, A478+F639, S488+S493, S488+T494, S488+T528, S488+V534, S488+M553, S488+L571, S488+I576, S488+V585, S488+A629, S488+V638, S488+F639, S493+T494, S493+T528, S493+V534, S493+M553, S493+L571, S493+I576, S493+V585, S493+A629, S493+V638, S493+F639, T494+T528, T494+V534, T494+M553, T494+L571, T494+I576, T494+V585, T494+A629, T494+V638, T494+F639, T528+V534, T528+M553, T528+L571, T528+I576, T528+V585, T528+A629, T528+V638, T528+F639, V534+M553, V534+L571, V534+I576, V534+V585, V534+A629, V534+V638, V534+F639, M553+L571, M553+I576, M553+V585, M553+A629, M553+V638, M553+F639, L571+I576, L571+V585, L571+A629, L571+V638, L571+F639, I576+V585, I576+A629, I576+V638, I576+F639, V585+A629, V585+V638, V585+F639, A629+V638, A629+F639, and V638+F639.

[0138] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid positions when compared to SEQ ID NO:2, wherein the amino acid positions are selected from the group consisting of E101+M156, E101+R180, E101+F233, E101+Q449, E101+D260, E101+K270, E101+Y273, E101+M367, E101+M553, E101+L571, E101+V585, E101+F639, M156+R180, M156+F233, M156+Q449, M156+D260, M156+K270, M156+Y273, M156+M367, M156+M553, M156+L571, M156+V585, M156+F639, R180+F233, R180+Q449, R180+D260, R180+K270, R180+Y273, R180+M367, R180+M553, R180+L571, R180+V585, R180+F639, F233+Q449, F233+D260, F233+K270, F233+Y273, F233+M367, F233+M553, F233+L571, F233+V585, F233+F639, Q449+D260, Q449+K270, Q449+Y273, Q449+M367, Q449+M553, Q449+L571, Q449+V585, Q449+F639, D260+K270, D260+Y273, D260+M367, D260+M553, D260+L571, D260+V585, D260+F639, K270+Y273, K270+M367, K270+M553, K270+L571, K270+V585, K270+F639, Y273+M367, Y273+M553, Y273+L571, Y273+V585, Y273+F639, M367+M553, M367+L571, M367+V585, M367+F639, M553+L571, M553+V585, M553+F639, L571+V585, L571+F639 and V585+F639. In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid positions when compared to SEQ ID NO:2, wherein said at least two amino acid positions are selected from the group consisting D260+R180, D260+M553, D260+Y273, R180+M553, R180+Y273, and M553+Y273. In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid position compared to SEQ ID NO:2, wherein said at least two amino acid substitutions are selected from the group consisting of M156I+R180G, M156I+F233M, M156I+D260G, M156I+D260S, M156I+K270R, M156I+Y273F, M156I+M367L, M156I+Q449N, M156I+M553A, M156I+L571S, M156I+V585R, M156I+V585D, M156I+F639V, R180G+F233M, R180G+D260G, R180G+D260S, R180G+K270R, R180G+Y273F, R180G+M367L, R180G+Q449N, R180G+M553A, R180G+L571S, R180G+V585R, R180G+V585D, R180G+F639V, F233M+D260G, F233M+D260S, F233M+K270R, F233M+Y273F, F233M+M367L, F233M+Q449N, F233M+M553A, F233M+L571S, F233M+V585R, F233M+V585D, F233M+F639V, D260G+D260S, D260G+K270R, D260G+Y273F, D260G+M367L, D260G+Q449N, D260G+M553A,D260G+L571S, D260G+V585R, D260G+V585D, D260G+F639V, D260S+K270R, D260S+Y273F, D260S+M367L, D260S+Q449N, D260S+M553A, D260S+L571S, D260S+V585R, D260S+V585D, D260S+F639V, Y273F+M367L, Y273F+Q449N, Y273F+M553A, Y273F+L571S, Y273F+V585R, Y273F+V585D, Y273F+F639V, M367L+Q449N, M367L+M553A, M367L+L571S, M367L+V585R, M367L+V585D, M367L+F639V, Q449N+M553A, Q449N+L571S, Q449N+V585R, Q449N+V585D, Q449N+F639V, M553A+L571S, M553A+V585R, M553A+V585D M553A+F639V, L571S+V585R, L571S+V585D, L571S+F639V, V585D+F639V and V585R+F639V.

[0139] In preferred embodiments the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid position compared to SEQ ID NO: 2, wherein said at least two amino acid substitutions are selected from the group consisting of D260N+R180G, D260S+R180G, D260S+M553A, D260S+Y273F, R180G+M553A, R180G+Y273F, M553A+Y273F, L571S+V585R, and L571S+V585D. Preferably, said at least two amino acid substitutions are selected from the group consisting of D260S+R180G M553A+Y273F, D260S+M553A, L571S+V585R, and L571S+V585D. More preferably, said at least two amino acid substitutions are selected from the group consisting of L571S+V585R, and L571S+V585D.

[0140] In a further preferred embodiment variant polypeptide with alpha-amylase activity according to the invention may advantageously comprise at least one further amino acid substitution in addition to said at least one or at least two amino acid substitutions in the position K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and / or F639 of SEQ ID NO: 1.

[0141] In a further preferred embodiment variant polypeptide with alpha-amylase activity according to the invention may advantageously comprise at least one further amino acid substitution in addition to said at least one or at least two amino acid substitutions in the position T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and / or F639 of SEQ ID NO: 1 or of SEQ ID NO:2.

[0142] In preferred embodiments of the variant polypeptide with alpha-amylase activity, said at least one further amino acid substitution occurs in a position selected from the group consisting of Y273,T68, D85, R95, 1125, P130, F136, S 141, T142, N151, A163, R180, E182, D198, L199, Q201, H232, F233, S235, Y258, G259, D260, D285, M367, F427, Q449, S488, S493, T494, T528, V534, M553 and A629 of SEQ ID NO: 1 or of SEQ ID NO:2.

[0143] In preferred embodiments of the variant polypeptide with alpha-amylase activity, said further amino acid substitution is selected from the group consisting of Y273F, T68A, D85G, R95S, R95H, I125C, I125Y, P130T, F136Y, S141A, T142S, T142G, N151D, A163S, R180G, R180D, R180Y, R180C, E182N, E182C, D198S, D198L, L199I, Q201V, Q201D, Q201C, H232D, F233M, S235E, S235A, S235F, S235M, Y258F, Y258A, Y258V, G259T, G259M, G259A, G259Y, G259V, G259S, G259H, D260G, D260S, D285R, M367L, F427G, F427A, Q449N, S488G, S493T, T494A, T528S, V534T, M553A, A629S and A629C.

[0144] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position Y273, wherein the substitution is selected from the group consisting ofY273F, Y273A, Y273R, Y273N, Y273D, Y273C, Y273Q, Y273E, Y273G, Y273H, Y273I, Y273L, Y273K, Y273M, Y273P, Y273S, Y273T, Y273W, and Y273V; preferably the substitution is Y273F.

[0145] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position T68, wherein the substitution is selected from the group consisting of T68A, T68R, T68N, T68D, T68C, T68Q, T68E, T68G, T68H, T68I, T68L, T68K, T68M, T68F, T68P, T68S, T68W, T68Y, and T68V; preferably the substitution is T68A.

[0146] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position D85, wherein the substitution is selected from the group consisting of D85G, D85S, D85A, D85R, D85N, D85C, D85Q, D85E, D85H, D85I, D85L, D85K, D85M, D85F, D85P, D85T, D85W, D85Y, and D85V; preferably the substitution is D85G.

[0147] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position R95, wherein the substitution is selected from the group consisting of R95S, R95H, R95A, R95N, R95D, R95C, R95Q, R95E, R95G, R95I, R95L, R95K, R95M, R95F, R95P, R95T, R95W, R95Y, and R95V; preferably the substitution is selected between R95S and R95H.

[0148] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position 1125, wherein the substitution is selected from the group consisting of I125C, I125Y, I125A, I125R, I125N, I125D, I125Q, I125E, I125G, I125H, I125L, I125K, I125M, I125F, I125P, I125S, I125T, I125W, and I125V; preferably the substitution is selected between I125C and I125Y.

[0149] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position P130, wherein the substitution is selected from the group consisting of P130T, P130A, P130R, P130N. P130D, P130C, P130Q, P130E, P130G, P130H, P130I, P130L, P130K, P130M, P130F, P130S, P130W, P130Y, and P130V; preferably the substitution is P130T.

[0150] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position F136, wherein the substitution is selected from the group consisting of F136Y, F136A, F136R, F136N, F136D, F136C, F136Q, F136E, F136G, F136H, F136I, F136L, F136K, F136M, F136P, F136S, F136T, F136W, and F136V; preferably the substitution is F136Y.

[0151] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position S 141, wherein the substitution is selected from the group consisting of S 141 A, S141R, S141N, S141D, S141C, S141Q, S141E, S141G, S141H, S141I, S141L, S141K, S141M, S141F, S141P, S141T, S141W, S141Y, and S141V; preferably the substitution is S141A.

[0152] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position T142, wherein the substitution is selected from the group consisting of T142G, T142S, T142A, T142R, T142N, T142D, T142C, T142Q, T142E, T142H, T142I, T142L, T142K, T142M, T142F, T142P, T142W, T142Y, and T142V; preferably the substitution is selected between T142G and T142S.

[0153] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position N151, wherein the substitution is selected from the group consisting of N151D, N151A, N151R, N151C, N151Q, N151E, N151G, N151H, N151I, N151L, N151K, N151M, N151F, N151P, N151S, N151T, N151W, N151Y, and N151V; preferably the substitution is N151D.

[0154] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position A 163, wherein the substitution is selected from the group consisting of A 163 S, A 163R, A 163N, A163D, A163C, A163Q, A163E, A163G, A163H, A163I, A163L, A163K, A163M, A163F, A163P, A163T, A163W, A163Y, and A 163 V; preferably the substitution is A163S.

[0155] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position R180, wherein the substitution is selected from the group consisting of R180G, R180D, R180Y, R180C, R180A, R180N, R180Q, R180E, R180H, R180I, R180L, R180K, R180M, R180F, R180P, R180S, R180T, R180W, and R180V; preferably the substitution is selected from the group consisting of R180G, R180D, R180Y, and R180C.

[0156] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position E182, wherein the substitution is selected from the group consisting of E182N, E182C, E182A, E182R, E182D, E182Q, E182G, E182H, E182I, E182L, E182K, E182M, E182F, E182P, E182S, E182T, E182W, E182Y, and E182V; preferably the substitution is selected between E182N and E182C.

[0157] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted 1 in position D198, wherein the substitution is selected from the group consisting of D198S, D198L, D198A, D198R, D198N, D198C, D198Q, D198E, D198G, D198H, D198I, D198K, D198M, D198F, D198P, D198T, D198W, D198Y, and D198V; preferably the substitution is selected between D198S and D198L.

[0158] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position L199, wherein the substitution is selected from the group consisting of L199I, L199A, L199R, L199N, L199D, L199C, L199Q, L199E, L199G, L199H, L199K, L199M, L199F, L199P, L199S, L199T, L199W, L199Y, and L199V; preferably the substitution is L199I.

[0159] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position Q201, wherein the substitution is selected from the group consisting of Q201V, Q201D, Q201C, Q201A, Q201R, Q201N, Q201E, Q201G, Q201H, Q201I, Q201L, Q201K, Q201M, Q201F, Q201P, Q201S, Q201T, Q201W, and Q201Y; preferably the substitution is selected from the group consisting of Q201V, Q201D and Q201C.

[0160] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position H232, wherein the substitution is selected from the group consisting of H232D, H232A, H232R, H232N, H232C, H232Q, H232E, H232G, H232I, H232L, H232K, H232M, H232F, H232P, H232S, H232T, H232W, H232Y, and H232V; preferably the substitution is H232D.

[0161] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position F233, wherein the substitution is selected from the group consisting of F233M, F233A, F233R, F233N, F233D, F233C, F233Q, F233E, F233G, F233H, F233I, F233L, F233K, F233P, F233S, F233T, F233W, F233Y, and F233V; preferably the substitution is F233M.

[0162] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position S235, wherein the substitution is selected from the group consisting of S235E, S235A, S235F, S235M, S235R, S235N, S235D, S235C, S235Q, S235G, S235H, S235I, S235L, S235K, S235P, S235T, S235W, S235Y, and S235V; preferably the substitution is selected from the group consisting of S235E, S235A, S235F, and S235M.

[0163] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position Y258, wherein the substitution is selected from the group consisting ofY258F, Y258A, Y258V, Y258R, Y258N, Y258D, Y258C, Y258Q, Y258E, Y258G, Y258H, Y2581, Y258L, Y258K, Y258M, Y258P, Y258 S, Y258T, Y258W, and Y258Y; preferably the substitution is selected from the group consisting of Y258F, Y258A, and Y258V.

[0164] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position G259, wherein the substitution is selected from the group consisting of G259T, G259M, G259A, G259Y, G259V, G259S, G259H, G259R, G259N, G259D, G259C, G259Q, G259E, G259I, G259L, G259K, G259F, G259P, and G259W; preferably the substitution is selected from the group consisting of G259T, G259M, G259A, G259Y, G259V, G259S and G259H.

[0165] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position D260, wherein the substitution is selected from the group consisting of D260G, D260S, D260A, D260R, D260N, D260C, D260Q, D260E, D260H, D260I, D260L, D260K, D260M, D260F, D260P, D260T, D260W, D260Y, and D260V; preferably the substitution is selected between D260G and D260S.

[0166] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position D285, wherein the substitution is selected from the group consisting of D285R, D285A, D285N, D285C, D285Q, D285E, D285G, D285H, D285I, D285L, D285K, D285M, D285F, D285P, D285S, D285T, D285W, D285Y, and D285V; preferably the substitution is D285R.

[0167] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position M367, wherein the substitution is selected from the group consisting of M367L, M367A, M367R, M367N, M367D, M367C, M367Q, M367E, M367G, M367H, M367I, M367K, M367F, M367P, M367S, M367T, M367W, M367Y, and M367V; preferably the substitution is M367L.

[0168] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position F427, wherein the substitution is selected from the group consisting of F427G, F427A, F427R, F427N, F427D, F427C, F427Q, F427E, F427H, F427I, F427L, F427K, F427M, F427P, F427S, F427T, F427W, F427Y, and F427V; preferably the substitution is selected between F427G and F427A.

[0169] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position Q449, wherein the substitution is selected from the group consisting of Q449N, Q449A, Q449R, Q449D, Q449C, Q449E, Q449G, Q449H, Q449I, Q449L, Q449K, Q449M, Q449F, Q449P, Q449S, Q449T, Q449W, Q449Y, and Q449V; preferably the substitution is Q449N.

[0170] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position S488, wherein the substitution is selected from the group consisting of S488G, S488A, S488R, S488N, S488D, S488C, S488Q, S488E, S488H, S488I, S488L, S488K, S488M, S488F, S488P, S488T, S488W, S488Y, and S488V; preferably the substitution is S488G.

[0171] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position S493, wherein the substitution is selected from the group consisting of S493T, S493A, S493R, S493N, S493D, S493C, S493Q, S493E, S493G, S493H, S493I, S493L, S493K, S493M, S493F, S493P, S493W, S493Y, and S493V; preferably the substitution is S493T.

[0172] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position T494, wherein the substitution is selected from the group consisting of T494A, T494R, T494N, T494D, T494C, T494Q, T494E, T494G, T494H, T494I, T494L, T494K, T494M, T494F, T494P, T494S, T494W, T494Y, and T494V; preferably the substitution is T494A.

[0173] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position T528, wherein the substitution is selected from the group consisting of T528S, T528A, T528R, T528N, T528D, T528C, T528Q, T528E, T528G, T528H, T528I, T528L, T528K, T528M, T528F, T528P, T528W, T528Y, and T528V; preferably the substitution is T528S.

[0174] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position V534, wherein the substitution is selected from the group consisting of V534T, V534A, V534R, V534N, V534D, V534C, V534Q, V534E, V534G, V534H, V534I, V534L, V534K, V534M, V534F, V534P, V534S, V534W, and V534Y; preferably the substitution is V534T.

[0175] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted compared to SEQ ID NO: 1 in position M553, wherein the substitution is selected from the group consisting of M553A, M553R, M553N, M553D, M553C, M553Q, M553E, M553G, M553H, M553I, M553L, M553K, M553F, M553P, M553S, M553T, M553W, M553Y, and M553V; preferably the substitution is M553A.

[0176] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, the amino acid sequence is substituted in position A629, wherein the substitution is selected from the group consisting of A629S, A629C, A629R, A629N, A629D, A629Q, A629E, A629G, A629H, A629I, A629L, A629K, A629M, A629F, A629P, A629T, A629W, A629Y, and A629V; preferably the substitution is selected between A629S and A629C.

[0177] In preferred embodiments of the variant polypeptide having alpha-amylase activity according to the invention, said further amino acid substitutions are selected from the group consisting ofY273F, R180G, F233M, D260S, M367L, Q449N, S493N, M553A. Even more preferably said amino acid substitutions are selected from the group consisting ofY273F, R180G, D260S, M553A. In a most preferred embodiment of the present invention, said further amino acid substitution is Y273F.

[0178] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid substitution in at least two amino acid position compared to SEQ ID NO: 1, wherein said at least two amino acid substitutions are selected from the group consisting of K270R, E101D, M156I, L571S, V585R, V585D, and F639V.

[0179] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention may comprise at least one further amino acid substitution in combination with one amino acid substitution occurring in position K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and / or F639 of SEQ ID NO: 1, selected from the group consisting of K270R+Y273F, K270R+M367L, K270R+Q449N, K270R+M553A, M156I+R180G, M156I+F233M, M156I+D260G, M156I+D260S, M156I+Y273F, M156I+M367L, M156I+Q449N, M156I+M553A, R180G+K270R, R180G+L571S, R180G+V585R, R180G+V585D, R180G+F639V, F233M+K270R, F233M+L571S, F233M+V585R, F233M+V585D, F233M+F639V, D260G+K270R, D260G+L571S, D260G+V585R, D260G+V585D, D260G+F639V, D260S+K270R, D260S+L571S, D260S+V585R, D260S+V585D, D260S+F639V, Y273F+L571S, Y273F+V585R, Y273F+V585D, Y273F+F639V, M367L+L571S, M367L+V585R, M367L+V585D, M367L+F639V, Q449N+L571S, Q449N+V585R, Q449N+V585D, Q449N+F639V, M553A+L571S, M553A+V585R, M553A+V585D, M553A+F639V. More preferably said combination of amino acid substitution is selected from the group consisting of D260S+K270R, R180G+K270R, M553A+K270R, K270R+Y273F,

[0180] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention may comprise at least one further amino acid substitution in combination with one amino acid substitution occurring in position T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and / or F639 of SEQ ID NO:2, selected from the group consistingof M156I+R180G, M156I+F233M, M156I+D260G, M156I+D260S, M156I+Y273F, M156I+M367L, M156I+Q449N, M156I+M553A, R180G+L571S, R180G+V585R, R180G+V585D, R180G+F639V, F233M+L571S, F233M+V585R, F233M+V585D, F233M+F639V, D260G+L571S, D260G+V585R, D260G+V585D, D260G+F639V, D260S+L571S, D260S+V585R, D260S+V585D, D260S+F639V, Y273F+L571S, Y273F+V585R, Y273F+V585D, Y273F+F639V, M367L+L571S, M367L+V585R, M367L+V585D, M367L+F639V, Q449N+L571S, Q449N+V585R, Q449N+V585D, Q449N+F639V, M553A+L571S, M553A+V585R, M553A+V585D, M553A+F639V. In a further preferred embodiment said further amino acid substitution - in addition to the amino acid substitution K270R already present in SEQ ID NO:2 - is selected from the group consisting of Y273F, D260S, R180G, M553A, Q449N M367L, and Y273F, preferably Y273F, D260S and M553A.

[0181] Functional characteristics of the variant polypeptide having alpha amylase activity according to the invention:

[0182] In preferred embodiments, the variant polypeptide according to the invention shows a malto-genic alpha-amylase activity.

[0183] In preferred embodiments, the variant polypeptide having an alpha-amylase activity according to the invention is capable of hydrolyzing (1→4)-α-d-glycosidic linkages in a starch or a related glucose polymer into malto-oligosaccharides.

[0184] In preferred embodiments, the variant polypeptide having an alpha-amylase activity according to the invention can hydrolize polymers comprising glucose molecules linked by alpha- 1,4-glycosidic bonds selected from amylose, amylopectin, dextrins, glycogen, and partially-hydrolyzed products thereof. Preferably, the variant polypeptide having an alpha-amylase activity according to the invention can hydrolize amylose, amylopectin, dextrins. More preferably, the variant polypeptide having an alphaamylase activity according to the invention can hydrolize amylose and amylopectin.

[0185] In preferred embodiments, the variant polypeptide having an alpha-amylase activity according to the invention can hydrolize a starch source selected from wheat flour, rice flour, com flour (maize flour), barley flour, oat flour, potato flour, cassava flour, tapioca flour, chickpea flour, almond flour,coconut flour, sorghum flour, and mixtures thereof. Preferably, the variant polypeptide having an alphaamylase activity according to the invention can hydrolize wheat flour.

[0186] In preferred embodiments, the variant polypeptide having an alpha-amylase activity according to the invention can hydrolize a starch source selected from a soluble starch, a resistant starch, or a damaged starch.

[0187] In preferred embodiments, the variant polypeptide having an alpha-amylase activity according to the invention leads to the formation of malto-oligosaccharides selected from the group consisting of maltose, maltotriose, maltotetraose, and higher oligosaccharides. Preferably, the variant polypeptide having an alpha-amylase activity according to the invention leads to the formation of maltose. More preferably, the variant polypeptide having an alpha-amylase activity leads to the formation of essentially maltose.

[0188] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention has at least one altered feature in comparison with SEQ ID NO: 1.

[0189] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits at least one altered feature in comparison to SEQ ID NO: 1 when added to a reaction media at a concentration within the ranges of 0.005 to 0.01, 0.01 to 0.05, 0.05 to 0.1, 0.1 to 0.3, 0.3 to 1.0, 1.0 to 3.0, 3.0 to 10.0, 10.0 to 30.0, 30.0 to 60.0, and 60.0 to 100.0 or any combination of these intervals.

[0190] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention has at least one altered feature in comparison with SEQ ID NO: 1, wherein the altered feature is selected from the group consisting of increased sucrose tolerance, increased enzymatic activity, increased activity on raw starch, and altered substrate specificity or a combination thereof.

[0191] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention, exhibits at least one altered feature in comparison with SEQ ID NO: 1, wherein the altered feature is selected from:a) an increased enzymatic activity compared to the alpha-amylase of SEQ ID NO: 1 under standard test conditions; and / orb) an increased sucrose-tolerance compared the alpha-amylase of SEQ ID NO: 1 under standard test conditions.

[0192] For the purposes of the present disclosure, "standard test conditions" refer to well-established methodologies and environmental parameters that are routinely recognized and utilized by those skilled in the art. These conditions typically include, but are not limited to, specific temperature ranges, humidity levels, pressure settings, and standardized testing protocols that ensure consistency and reliability of results. The techniques and conditions described herein are provided as illustrative examples. However, a skilled person in the field will appreciate how to modify and adapt these techniques and conditions based on specific requirements or varying circumstances.

[0193] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits an altered enzymatic activity, which is increased when compared to the alphaamylase of SEQ ID NO: 1 under standard test conditions.

[0194] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits an increased enzymatic activity compared to SEQ ID NO: 1, wherein the increase in enzymatic activity ranges from about 0.1% to about 300% or more relative to the alpha-amylase of SEQ ID NO: 1. Preferably, the increase in enzymatic activity is at least 0.1 %, preferably of at least 0.2 %, more preferably at least 10 %, still more preferably at least 30 %, yet more preferably at least 40 % relative to the alpha-amylase of SEQ ID NO: 1.

[0195] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits an increased enzymatic activity compared to SEQ ID NO: 1, wherein the increase in enzymatic activity ranges from at least 1.1 -fold to 6-fold relative to the alpha-amylase of SEQ ID NO: 1. Preferably, the increase in enzymatic activity is at least 1.1-fold, preferably at least 1.2-fold, more preferably at least 1.3-fold, still more preferably at least 1.4-fold, yet more preferably at least 1.5-fold, even more preferably at least 2.0-fold, and most preferably at least 2.5-fold relative to the alphaamylase of SEQ ID NO: 1.

[0196] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits an increased sucrose tolerance when compared to the alpha-amylase of SEQ ID NO: 1 under standard test conditions.

[0197] In preferred embodiments, the sucrose-tolerance of the variant polypeptide having alpha-amylase activity according to the invention is represented by an altered enzymatic activity in the presence of sucrose, which is increased when compared to the alpha-amylase of SEQ ID NO: 1 under standard test conditions.

[0198] In preferred embodiments, the enzymatic activity in the presence of sucrose of the variant polypeptide having alpha-amylase activity according to the invention is determined at a sucrose concentration ranging from about 15 mM to about 300 mM, preferably from about 15 mM to about 150 mM, more preferably from about 30 mM to about 150 mM, more preferably from about 60 mM to about 150 mM, more preferably from about 90 mM to about 150 mM, still more preferably from about 120 mM to about 150 mM. Preferably, the enzymatic activity in the presence of sucrose of the variant polypeptide having alpha-amylase activity according to the invention is determined at a sucrose concentration of at least 15 mM, preferably at least 30 mM, more preferably at least 60 mM, still more preferably at least 90 mM, even most preferably at least 120 mM, and utmost preferably at least 150 mM.

[0199] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits an increased sucrose tolerance compared to SEQ ID NO: 1, wherein the increase in sucrose tolerance ranges from about 0.1% to about 600% or more relative to the alpha -amylase of SEQ ID NO: 1. Preferably, the increase in sucrose tolerance is at least 10 %, preferably of at least 20 %, more preferably at least 30 %, still more preferably at least 40 %, yet more preferably at least 50 % relative to the alpha-amylase of SEQ ID NO: 1.

[0200] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits an increased sucrose tolerance compared to SEQ ID NO: 1, wherein the increasein sucrose tolerance ranges from at least 1.1 -fold to 6-fold relative to the alpha-amylase of SEQ ID NO: 1. Preferably, the increase in sucrose tolerance is at least 1.1 -fold, preferably at least 1.2-fold, more preferably at least 1.3-fold, still more preferably at least 1.4-fold, yet more preferably at least 1.5-fold, even more preferably at least 2.0-fold, and most preferably at least 2.5 -fold relative to the alpha-amylase of SEQ ID NO: 1.

[0201] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1, wherein the variant polypeptide having alpha-amylase activity has at least one of altered features (A) and (B), or a combination thereof; wherein(A) is an increase in enzymatic activity of at least 1.1 -fold relative to the alpha-amylase of SEQ ID NO 1 under standard test conditions; and(B) is an increased sucrose tolerance, wherein the sucrose tolerance is determined as enzymatic activity in the presence of sucrose, wherein the enzymatic activity in the presence of sucrose is at least 1.1-fold higher than the enzymatic activity in the presence of sucrose of the alpha-amylase of SEQ ID NO: 1 under standard test conditions.

[0202] In preferred embodiments, the variant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid amino acid position compared to SEQ ID NO: 1, wherein the amino acid position is selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638, and F639; wherein the variant polypeptide having alpha-amylase activity has at least one of altered features (A) and (B), or a combination thereof; wherein(A) is an increase in enzymatic activity of at least 1.1 -fold relative to the alpha-amylase of SEQ ID NO 1 under standard test conditions; and(B) is an increased sucrose tolerance, wherein the sucrose tolerance is determined as enzymatic activity in the presence of sucrose, wherein the enzymatic activity in the presence of sucrose is at least 1.1-fold higher than the enzymatic activity in the presence of sucrose of the alpha-amylase of SEQ ID NO: 1 under standard test conditions.

[0203] In preferred embodiments, the variant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid amino acid position compared to SEQ ID NO: 1, wherein the amino acid position is selected from the group consisting of:a) K270, E101, M156, 3L571, V585, and F639; orb) K270, L57I, V585, 3and F639;wherein the variant polypeptide having alpha-amylase activity has at least one of altered features (A) and (B), or a combination thereof; wherein(A) is an increase in enzymatic activity of at least 1.1 -fold relative to the alpha-amylase of SEQ ID NO 1 under standard test conditions; and(B) is an increased sucrose tolerance, wherein the sucrose tolerance is determined as enzymatic activity in the presence of sucrose, wherein the enzymatic activity in the presence of sucrose is at least 1.1-fold higher than the enzymatic activity in the presence of sucrose of the alpha-amylase of SEQ ID NO: 1 under standard test conditions.

[0204] In preferred embodiments, the variant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one position compared to SEQ ID NO: 1, wherein the amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A, Q99R, E101D, A138VY155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, A478S, L571G, L571S, I576L, I576V, V585R, V585D, V638D, and F639V; wherein the variant polypeptide having alpha-amylase activity has at least one of altered features (A) and (B), or a combination thereof; wherein(A) is an increase in enzymatic activity of at least 1.1 -fold relative to the alpha-amylase of SEQ ID NO 1 under standard test conditions; and(B) is an increased sucrose tolerance, wherein the sucrose tolerance is determined as enzymatic activity in the presence of sucrose, wherein the enzymatic activity in the presence of sucrose is at least 1.1-fold higher than the enzymatic activity in the presence of sucrose of the alpha-amylase of SEQ ID NO: 1 under standard test conditions.

[0205] In preferred embodiments, the variant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid amino acid position compared to SEQ ID NO: 1, wherein the amino acid position is selected from the group consisting of:a) K270R, E101D, MI56I, L571S, V585R, and F639V;b) K270R, E101D, MI56I, L571S, V585D, and F639V:c) K270R, L571S, V585R, and F639V; ord) K270R, L571S, V585D, and F639V.wherein the variant polypeptide having alpha-amylase activity has at least one of altered features (A) and (B), or a combination thereof; wherein(A) is an increase in enzymatic activity of at least 1.1 -fold relative to the alpha-amylase of SEQ ID NO 1 under standard test conditions; and(B) is an increased sucrose tolerance, wherein the sucrose tolerance is determined as enzymatic activity in the presence of sucrose, wherein the enzymatic activity in the presence of sucrose is at least 1.1-fold higher than the enzymatic activity in the presence of sucrose of the alpha-amylase of SEQ ID NO: 1 under standard test conditions.

[0206] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits at least one altered feature in comparison with SEQ ID NO: 1, wherein the alteredfeature is one property generally pertaining to the utilization of an alpha-amylase in the creation of doughs and baked products.

[0207] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention has at least one altered feature in comparison with SEQ ID NO: 1, wherein the altered feature is selected from the group consisting of reduced hardness after storage of a baked product, reduced loss of resilience over storage of a baked product, improved springiness of the backed product, improved cohesiveness of the baked product, improved water mobility of the baked product, increased strength of the dough, increased elasticity of the dough, increased stability of the dough, reduced stickiness of the dough, improved extensibility of the dough, improved machineability of the dough, increased volume of the baked product, improved flavor of the baked product, improved crumb structure of the baked product, improved crumb softness of the baked product, reduced blistering of the baked product, improved crispiness of the baked product, improved resilience both initially and after storage, improved anti-staling of the baked product, faster dough development time, reduced dough stickiness, improved foldability of the baked product, improved flexibility of the baked product, improved stackability of flat baked products, increased flexibility of noodles, reduced clumping of cooked noodles, improved flavor retention of noodles, reduction of hairline cracks in crackers, enhanced leavening effect, improved mouthfeel, improved softness on squeeze, reduced damage during transport, reduced breaking during transport, improved resilience of the bake product, reduced loss of resilience over storage of the baked product, and improved sliceability.

[0208] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention, in comparison with SEQ ID NO: 1, leads to at least one altered feature related to the texture of a dough or baked product, wherein the altered feature is selected from the group consisting of reduced hardness after storage of the baked product, improved springiness of the baked product, reduced loss of resilience over storage of the baked product, improved cohesiveness of the baked product, and improved water mobility of the baked product.

[0209] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention, in comparison with SEQ ID NO: 1, leads to at least one altered feature related to thesensory perception of a dough or baked product, wherein the altered feature is selected from the group consisting of Softness to touch, Softness to eat, Firmness, Moistness, and Preferability.

[0210] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention, in comparison with SEQ ID NO: 1, leads to at least one altered feature to a dough or baked product, wherein the dough or baked product comprises:a) sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight; orb) sugar and / or sucrose in an amount ranging from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

[0211] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits a hardness after storage of the baked product that compared to SEQ ID NO: 1 is reduced from 1.051-fold to 3-fold, preferably from 1,1-fold to 2.5-fold; more preferably from 1.5-fold to 2.3-fold, or any value or any value within or between these ranges.

[0212] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits a springiness after storage of the baked product that compared to SEQ ID NO: 1 is improved from 1.051 -fold to 3 -fold, preferably from 1,1 -fold to 2.5 -fold; more preferably from 1.5-fold to 2.3-fold, or any value or any value within or between these ranges.

[0213] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits a loss of resilience over storage of the baked product that compared to SEQ ID NO:1 is reduced from 1.051-fold to 3-fold, preferably from 1,1-fold to 2.5-fold; more preferably from 1.5-fold to 2.3-fold, or any value or any value within or between these ranges.

[0214] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits a cohesiveness of the baked product that compared to SEQ ID NO: 1 is improved from 1.051-fold to 3-fold, preferably from 1,1-fold to 2.5-fold; more preferably from 1.5-fold to 2.3-fold, or any value or any value within or between these ranges.

[0215] In preferred embodiments, the variant polypeptide having alpha-amylase activity according to the invention exhibits a water mobility of the baked product that compared to SEQ ID NO: 1 is improved from 1.051-fold to 3-fold, preferably from 1,1-fold to 2.5-fold; more preferably from 1.5-fold to 2.3-fold, or any value or any value within or between these ranges.

[0216] Second aspect: expression method:

[0217] A further aspect of the invention relates to a method of expressing a variant polypeptide having alpha amylase activity according to the invention comprising:a) cultivating a recombinant host cell under conditions appropriated to production of a variant polypeptide having alpha amylase activity according to the invention; andb) recovering a variant polypeptide having alpha amylase activity according to the invention.

[0218] In preferred embodiments, the recombinant host cell used in step (a) of the method according to the invention is a recombinant bacterial host cell.

[0219] In preferred embodiments, the recombinant bacterial host cell is selected from a gram-positive bacterial species or from a gram-negative species.

[0220] In preferred embodiments, the recombinant bacterial host cell is a gram-positive bacterial species selected from the group consisting of the following species: Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus thuringiensis, Bacillus anthracis, Lactococcus lactis, Streptococcus spp., Streptomyces lividans, Streptomyces coelicolor, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Lactobacillus spp., Staphylococcus spp. (e.g., Staphylococcus carnosus), Clostridium spp. (e.g., Clostridium acetobutylicum), Enterococcus faecalis, Propi-onibacterium spp., Mycobacterium spp. (e.g., Mycobacterium smegmatis), Actinomyces spp., Micrococcus spp., and Rhodococcus spp. Preferably, the recombinant bacterial host cell is a Bacillus subtilis recombinant cell.

[0221] In preferred embodiments, the recombinant bacterial host cell is a gram-negative bacterial species selected from the group consisting of the following species: Escherichia coli, Pseudomonas putida,Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae, Acinetobacter baylyi, Agrobacterium tumefaciens, Burkholderia cepacia, Yersinia enterocolitica, Vibrio cholerae, Shigella flexneri, Neisseria meningitidis, Haemophilus influenzae, Legionella pneumophila, Bordetella pertussis, Helicobacter pylori, Proteus mirabilis, Francisella tularensis, Brucella spp., Rickettsia spp., Moraxella spp., Campylobacter jejuni, Borrelia burgdorferi, and Coxiella burnetii. Preferably, the recombinant bacterial host cell is an Escherichia coli recombinant cell.

[0222] In preferred embodiments, the recombinant host cell used in step (a) of the method according to the invention comprises a recombinant expression vector comprising a nucleic acid construct which comprises a nucleic acid encoding an amino acid sequence of a variant polypeptide having alpha-amylase activity according to the invention operably linked to one or more control sequences capable of directing the expression of an alpha-amylase in a suitable expression host.

[0223] In preferred embodiments, the nucleic acid used in the method according to the invention encodes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.

[0224] In preferred embodiments, the nucleic acid sequence according to the invention encodes an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid position is selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638, and F639.

[0225] In preferred embodiments, the nucleic acid sequence according to the invention encodes an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A, Q99R, E101D, A138V, Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, A478S, L571G, L571S, I576L, I576V, V585R, V585D, V638D, and F639V.

[0226] In preferred embodiments, the nucleic acid sequence used in the method according to the invention encodes a variant polypeptide having alpha-amylase activity comprising an amino acid sequence encompassed by any of the preferred embodiments according to the first aspect of the invention.

[0227] Third aspect – enzyme compositions:

[0228] One further aspect of the present disclosure is to provide a composition comprising a variant polypeptide having alpha-amylase activity according to the invention and one or more components.

[0229] In preferred embodiments, the composition according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.

[0230] In preferred embodiments, the composition according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid position is selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265 N309, K318, A368, A478, L571, 1576, V585, V638, and F639.

[0231] In preferred embodiments, the composition according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A, Q99R, E101D, A138V, Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, A478S, L571G, L571S, I576L, I576V, V585R V585D, V638D, and F639V.

[0232] In preferred embodiments, the composition according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence encompassed by any of thepreferred embodiments according to the first aspect of the invention or obtained by a method of any of the preferred embodiments according to the second aspect of the invention.

[0233] In one preferred embodiment, the composition according to the invention is a dough conditioner composition.

[0234] In a preferred embodiment, the dough conditioner composition comprising the variant polypeptide having alpha-amylase activity according to the invention is provided in a dry form or liquid forms, but preferably in dry form. A dry form includes, but is not limited to, powders, granules, or flakes. A liquid form includes, but is not limited to, an emulsion, a suspension, and a solution. Regardless of the constitution of the dough conditioner composition, any additive or additives recognized for their efficacy in enhancing and / or sustaining the enzyme's effectiveness, the dough's quality, and / or the final baked product may be employed.

[0235] In preferred embodiments, the dough conditioner composition according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence encompassed by any of the preferred embodiments according to the first aspect of the invention or obtained by a method of any of the preferred embodiments according to the second aspect of the invention, and at least one additional component.

[0236] In preferred embodiments, the dough conditioner composition comprises the variant polypeptide having alpha-amylase activity according to the invention and one or more components selected from the group consisting of diluents, additional enzymes, emulsifiers, oxidizing agents, reducing agents, hydrocolloids, surfactants, dough conditioner agents, flavor enhancers, preservatives, and nutritional supplements.

[0237] Examples of diluents include, but are not limited to, milk powder, gluten, granulated fat, maltodextrin, cornstarch, rice flour, wheat flour, soy flour, oat flour, potato starch, tapioca starch, cellulose, and powdered sugar.

[0238] Examples of additional enzymes include, but are not limited to, a further alpha-amylase (such as a fungal alpha-amylase, a beta-amylase, a cyclodextrin glucotransferase, a protease, a peptidase, atransglutaminase, a lipase, a triacyl glycerol lipase, a galactolipase, a phospholipase, a cellulase, a hemicellulase, a protein disulfide isomerase, a glycosyltransferase, a peroxidase, a laccase, a hexose oxidase, an aldose oxidase, a pyranose oxidase, and a lipoxygenase.

[0239] Examples of emulsifiers include, but are not limited to, soy lecithin, mono- and diglycerides of fatty acids, polysorbate 80, sodium stearoyl lactylate, DATEM (Diacetyl Tartaric Acid Esters of Monoglycerides), lecithin, and glycerol monostearate.

[0240] Examples of oxidizing agents include, but are not limited to, potassium bromate, ascorbic acid (vitamin C), bromate salts, bromated flour, azodicarbonamide (ADA), and potassium iodate.

[0241] Examples of reducing agents include, but are not limited to, L-cysteine, sodium metabisulfite, glutathione, cysteamine hydrochloride, and erythorbic acid.

[0242] Examples of hydrocolloids include, but are not limited to, xanthan gum, guar gum, carrageenan, agar-agar, pectin, locust bean gum, and konjac gum.

[0243] Examples of surfactants include, but are not limited to, sodium caseinate, sorbitan monostearate, polysorbate 60, polysorbate 80, sodium stearoyl lactylate, sodium lauryl sulfate, and sorbitan tristearate.

[0244] Examples of dough conditioner agents include, but are not limited to, calcium carbonate, sodium acid pyrophosphate, potassium bromate, diacetyl tartaric acid esters of mono- and diglycerides (DATEM), and ammonium chloride.

[0245] Examples of flavor enhancers include, but are not limited to, monosodium glutamate (MSG), yeast extract, malt extract, hydrolyzed vegetable protein, and disodium inosinate.

[0246] Examples of preservatives include, but are not limited to, calcium propionate, sodium benzoate, potassium sorbate, natamycin, and propyl gallate.

[0247] Examples of nutritional supplements include, but are not limited to, vitamins (e.g., vitamin C, vitamin E, vitamin B complex), minerals (e.g., calcium carbonate, iron sulfate, zinc oxide), amino acids (e.g., lysine, methionine), omega-3 fatty acids, and fiber additives (e.g., inulin, psyllium husk).

[0248] In one preferred embodiment, the composition comprising the variant polypeptide according to the invention is a pre-mix composition.

[0249] In preferred embodiments, the pre-mix composition according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence encompassed by any of the preferred embodiments according to the first aspect of the invention or obtained by a method of any of the preferred embodiments according to the second aspect of the invention, and at least one additional component.

[0250] In a preferred embodiment, a pre-mix composition according to the invention comprises the variant polypeptide having alpha-amylase activity according to the invention, and flour.Fourth aspect – use of a variant polypeptide having alpha-amylase:

[0251] A further aspect of the invention refers to the use of a variant polypeptide having alpha-amylase activity according to the invention or of a composition according to the invention or of a pre -mix according to the invention in the preparation of a dough and / or a baked product.

[0252] Preferred embodiments according to the invention refer to the use of a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 in the preparation of a dough and / or a baked product.

[0253] Preferred embodiments according to the invention refer to the use of a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid position is selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, L571, 1576, V585, V638, and F639 in the preparation of a dough and / or a baked product.

[0254] Preferred embodiments according to the invention refer to the use of a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ IDNO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A, Q99R, E101D, A138V, Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, L571G, L571S, I576L, I576V, V585R, V585D, V638D, and F639V in the preparation of a dough and / or a baked product.

[0255] Preferred embodiments according to the invention refer to the use of a variant polypeptide having alpha-amylase activity comprising an amino acid sequence encompassed by any of the preferred embodiments according to the first aspect of the invention, or a variant polypeptide having alpha-amylase activity obtained by a method of any of the preferred embodiments according to the second aspect of the invention, or a composition comprising a variant polypeptide having alpha-amylase activity encompassed by any of the preferred embodiments according to the third aspect of the invention in the preparation of a dough and / or a baked product.

[0256] In a preferred embodiment, the use according to the invention is in the preparation of a dough and / or a baked product selected from the group consisting of bread, including white bread, brown bread, whole-meal bread, French baguette -style bread, pastries like croissants, brioche, tin bread, twisted bread, buns (such as hamburger buns or steamed buns), chapati, rusk, dried steam bun slices, bread crumbs, matzos, focaccia, melba toast, zwieback, croutons, soft pretzels, soft bread, hard bread, breadsticks, leavened breads (yeast and chemically-leavened), pita bread and other flatbreads, tortillas, tacos, steamed bread, crispbread, and panettone; laminated dough products, including Danish pastry and croissants; pasta, including noodles prepared by boiling or stir-frying; soft doughs including cakes, pancakes, cookies, biscuits, doughnuts (including yeasted doughnuts), bagels, pie crusts, brownies, sheet cakes; and snack foods, including pretzels, tortilla chips, fabricated snacks, and fabricated potato crisps.

[0257] In a preferred embodiment, the use according to the invention is in the preparation of a sugar-containing dough and / or baked product.

[0258] In a preferred embodiment, the use according to the invention is in the preparation of a sucrose-containing dough and / or baked product.

[0259] In a preferred embodiment, the use according to the invention is in the preparation of a dough and / or baked product comprising sucrose or sugar at a concentration of at least 5 wt%, at least 8 wt%, at least 12 wt%, at least 15 wt%, at least 18 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, or at least 40 wt% based on flour total weight. For the purposes of the present disclosure, sucrose weight amount is calculated as a function of the total weight of flour added in the recipe, for instance, to accomplish a dough comprising 5 wt% sucrose, 50 grams sucrose are added per 1000 gram of flour used in the recipe.

[0260] In a preferred embodiment, the use according to the invention is in the preparation of a dough and / or baked product comprising sucrose or sugar at a concentration of at least 10 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt% based on weight of total ingredients. Preferably, the dough or baked product comprises a sucrose content ranging from 10 wt% to 40 wt%, more preferably from 20 wt% to 35 wt% based on weight of total ingredients.Fifth aspect – dough / baked product:

[0261] A further aspect of the invention relates to a dough or a baked product comprising a variant polypeptide having alpha-amylase activity according to the invention, a dough conditioner composition according to the invention, or a pre-mix composition according to the invention.

[0262] In a preferred embodiment, the dough or the baked product according to the invention is a sugar-containing dough and / or baked product.

[0263] In a preferred embodiment, the dough or the baked product according to the invention is a sucrose -containing dough and / or baked product.

[0264] In preferred embodiments, the dough and / or baked product comprises sucrose or sugar at a concentration of at least 5 wt%, at least 8 wt%, at least 12 wt%, at least 15 wt%, at least 18 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, or at least 40 wt% based on flour total weight. For the purposes of the present disclosure, sucrose weight amount is calculated as a function of the total weight of flour added in the recipe, for instance, to accomplish a dough comprising 5 wt% sucrose, 50 grams sucrose are added per 1000 gram of flour used in the recipe.

[0265] In preferred embodiments, the dough and / or baked product comprises sucrose or sugar at a concentration of at least 10 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt% based on weight of total ingredients. Preferably, the dough or baked product comprises a sucrose content ranging from 10 wt% to 40 wt%, more preferably from 20 wt% to 35 wt% based on weight of total ingredients.

[0266] In preferred embodiments, the dough or the baked product according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1, and leads to at least one altered feature to a dough or baked product in comparison with SEQ ID NO: 1; and wherein the dough or baked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

[0267] In preferred embodiments, the dough or the baked product according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid position is selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, L571, 1576, V585, V638, and F639; and wherein the dough or baked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

[0268] In preferred embodiments, the dough or the baked product according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A, Q99R, E101D, A138V, Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, L571G, L571S, I576L, I576V, V585R, V585D, V638D, and F639V; and wherein the dough orbaked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

[0269] In preferred embodiments, the dough or the baked product according to the invention comprises a variant polypeptide having alpha-amylase activity comprising an amino acid sequence encompassed by any of the preferred embodiments according to the first aspect of the invention, or a variant polypeptide having alpha-amylase activity obtained by a method of any of the preferred embodiments according to the second aspect of the invention, or a composition comprising a variant polypeptide having alpha-amylase activity encompassed by any of the preferred embodiments according to the third aspect of the invention.Sixth aspect – method of preparing a dough or a baked product:

[0270] A further aspect of the invention relates to a method of preparing a dough or a baked product according to the invention comprising adding a variant polypeptide having alpha-amylase activity according to the invention to the dough or baked product.

[0271] In preferred embodiments, the method according to the invention comprises adding to a dough or baked product a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1, and leads to at least one altered feature to a dough or baked product in comparison with SEQ ID NO: 1;wherein the dough or baked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

[0272] In preferred embodiments, the method according to the invention comprises adding to a dough or baked product a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in atleast one amino acid position compared to SEQ ID NO: 1, wherein the amino acid position is selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, L571, 1576, V585, V638, and F639;wherein the dough or baked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

[0273] In preferred embodiments, the method according to the invention comprises adding to a dough or baked product a variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 1 and comprising an amino acid substitution in at least one amino acid position compared to SEQ ID NO: 1, wherein the amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A, Q99R, E101D, A138V, Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, L571G, L571S, I576L, I576V, V585R, V585D, V638D, and F639V; wherein the dough or baked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

[0274] In preferred embodiments, the method according to the invention comprises adding to a dough or backed product a variant polypeptide having alpha-amylase activity comprising an amino acid sequence encompassed by any of the preferred embodiments according to the first aspect of the invention, or a variant polypeptide having alpha-amylase activity obtained by a method of any of the preferred embodiments according to the second aspect of the invention, or a composition comprising a variant polypeptide having alpha-amylase activity encompassed by any of the preferred embodiments according to the third aspect of the invention, wherein the dough or backed product is encompassed by any of the preferred embodiments according to the fifth aspect of the invention.EXAMPLES

[0275] The following examples further illustrate the invention but are not to be construed as limiting its scope.Example 1. General methods

[0276] The gene for the maltogenic amylase was cloned into the Bacillus expression plasmid pLE2D06 fused with a suitable signal peptide for efficient secretion into the media. The secretion sequence of lytF showed high level of secreted maltogenic amylase. The vector pLE2D06 is induced based on the growth phase and contains a chloramphenicol resistance gene for selection as well. The resulting plasmid was used for transformation of Bacillus subtilis strain LE2A106 (a derivative of Bacillus subtilis DSM No.402).

[0277] Variants of the maltogenic amylase (Table 1) were created by standard site directed mutagenesis technologies as known to the person skilled in the art and were than recombinantly expressed.

[0278] Table 1: Variants of the alpha amylase of the invention (in bold) with one substitution in comparison with SEQ ID NO: 1 (wild-type, Geobacillus stearothermophilus)'Variant Variant Variantnumber 1stmutation number 1stmutation number 1stmutation 2 T25A 32 Q201V 62 K270I3 T68A 33 Q201D 63 K270L4 D85G 34 Q201C 64 K270F5 R95S 35 H232D 65 K270S6 R95H 36 F233M 66 Y273F7 Q99R 37 S235E 67 D285R8 E101D 38 S235A 68 N309T9 I125C 39 S235F 69 K318E10 I125Y 40 S235M 70 K318I11 P130T 41 Y258F 71 M367L 12 F136Y 42 Y258A 72 A368S13 A138V 43 Y258V 73 F427G14 S141A 44 G259T 74 F427A15 T142G 45 G259M 75 Q449N16 T142S 46 G259A 76 A478S17 N151D 47 G259Y 77 S488G18 Y155L 48 G259V 78 S493T19 M156I 49 G259S 79 T494A20 A163S 50 G259H 80 T528S21 R180G 51 D260G 81 V534T22 R180D 52 D260S 82 M553A23 R180Y 53 A265P 83 L571G24 R180C 54 A265S 84 L571S25 E182N55 K270R85 I576L26 E182C 56 K270V 86 I576V27 W185Y 57 K270G 87 V585R28 D198S 58 K270N 88 A629S29 D198L 59 K270D 89 A629C30 L199I 60 K270C 90 V638D61 K270H91 F639V

[0279] Recombinant maltogenic amylase enzymes were routinely expressed in 96-deep-well plates in LB medium (10 g / 1 trypton, 5 g / 1 yeast extract, 10 g / 1 NaCl, pH 7.0) with 10 pg / ml chloramphenicol and 1 mM MgSO4. For the cultivation of precultures the medium was supplemented with 2 % glucose, and 0.4 ml medium per well were inoculated with a single clone and incubated overnight at 37 °C and 1000 rpm for agitation. For expression cultures the medium was supplemented with 0.05 % glucose and 2 % maltose and 1 ml of medium per well was inoculated with 10 pl of a fresh overnight preculture. The cultures were incubated for 48 hours with shaking at 37 °C for expression and secretion of malto-genic amylase into the medium. The supernatants containing secreted maltogenic amylase were separated from cells by centrifugation (3300xg; 20 min; 4 °C) and the resulting enzyme samples were used for further analysis.Example 2, Maltogenic amylase activity assay:

[0280] The assay uses maltotriose as substrate. The maltogenic amylase hydrolyzes the substrate to yield glucose and maltose. The produced glucose is detected using the D-glucose HK Assay Kit (Megazymes K-GLUHK-220A) according to the kit manual. The assay is performed in 96-well plates.

[0281] Typically, the supernatant from expression cultures (Example 1) are diluted 1 part supernatant plus 1 part 0.1 M NaOAc buffer (pH 4.8) to adjust the pH, corresponding to a 2-fold dilution, and in a second step 1 part of the resulting enzyme with 9 parts of 0.1 M NaOAc buffer (pH 5.0), corresponding to a 10-fold dilution. The total dilution of the supernatant was typically a 20-fold dilution. A 96-well plate with 50 pl 2 % (w / w) maltotriose and 25 pL 0.1 M NaOAc buffer (pH 5.0) per well is preheated to 40 °C for 10 min before 25 pL of the diluted culture supernatant is added to yield a total volume of 100 pL per well. The biotransformation is incubated for 60 min at 40°C. The reaction is stopped by the addition of 125 pL of 2 M Tris-HCl buffer (pH 7.55). The concentration of released glucose by hydrolysis of maltotriose is measured with the D-glucose HK Assay Kit according to the microplate assay procedure from the manual. The absorbance is measured using a plate photometer at 340 nm. For thequantification a calibration curve is measured using eight glucose standard solutions with concentrations between 0 mM and 4 mM.

[0282] Table 2: Enzymatic activity of the single variants of the alpha amylase of the invention (in bold) in comparison with SEQ ID NO: 1 (=wild-type, Geobacillus stearothermophilus)' expressed as improvement factor (IF) over wild-type (IFWT = 1.0):VariVariVariVariIF IF IF IFant ant ant ant1 1.0 19 1.2 51-52 1.1 - 1.2 79 1.12 1.1 20 1.2 53-54 1.1 - 1.3 80 1.13 1.1 21 1.1 55 1.2 81 1.14 1.2 25 1.1 66 1.1 82 1.35 1.2 27 1.2 68 1.2 83 1.26 1.1 28 1.1 69-70 1.1 - 1.2 84 1.27 1.1 30 1.2 72 1.2 85 1.28 1.2 73 1.1 86 1.113 1.2 32 1.2 74 1.2 87 1.315 1.1 36 1.3 75 1.2 89 1.116 1.1 37 1.1 76 1.2 90 1.117 1.2 38 1.1 77 1.291 1.418 1.244-50 0.8 - 1.178 1.3Example 3, Sucrose tolerance assay:

[0283] This assay determines the effect of inhibition on the maltogenic amylase and its variants by the presence of 150 mM sucrose. It is performed as described in Example 2 but in presence of 150 mM sucrose. Therefore 25 pL of a 600 mM sucrose solution in 0.1 M NaOAc buffer (pH 5.0) are added instead of the buffer without sucrose from Example 2. Since the activity is reduced compared to the activity without sucrose, the total dilution of the supernatant from Example 1 was reduced to 1:10.

[0284] Table 3: Enzymatic activity in the presence of sucrose (sucrose tolerance) of the single variants according to the invention (in bold) in comparison with SEQ ID NO: 1 (=wild-type, Geobacillus stea-rothermophilus) expressed as improvement factor (IF) over wild-type (IFWT = 1.0):VariVariVariVariIF IF IF IFant ant ant ant1 1.0 19 1.4 37 - 40 1.2 - 1.3 78 1.32 1.2 20 1.4 44 - 50 1.4 - 1.6 79 1.33 1.3 21 2.0 51 - 52 1.4 - 2.2 80 1.14 1.5 22 1.8 53 - 54 1.3 - 1.4 81 1.25 1.523 1.655 2.482 1.66 1.2 24 1.5 66 1.2 83 1.57 1.2 25 1.3 67 1.2 84 1.58 1.5 26 1.1 68 1.3 85 1.211 1.3 27 1.3 69 - 70 1.2 86 1.112 1.2 28 1.4 71 1.7 87 1.613 1.3 30 1.5 72 1.3 88 1.114 1.2 73 1.3 89 1.115 1.1 32 1.4 74 1.1 90 1.216 1.1 33 1.2 75 1.391 1.717 1.4 34 1.1 76 1.218 1.136 1.577 1.2Example 4: Performance evaluation of baked dough comprising the variant polypeptide having alphaamylase activity of the invention

[0285] Doughs having 5 wt% - 40 wt% of flour (based on the total dough / batter weight) will be made with addition of amylase as follows: amylase according to the invention in different dosages, e.g. 1, 10, 100 or 1000 mg protein enzyme / kg flour. Comparators and control doughs without maltogenic amylase and / or comprising the amylase of SEQ ID NO: 1 and / or comprising a commercial amylase may be included. Doughs may include bread and / or white cake or any other dough or batter cited in this specification.

[0286] Doughs will be prepared using laboratory-scale recipe and baking procedure (same base formula, mixing time, fermentation, proofing and baking times and temperatures). The dough will be baked and cooled down for 60-120 minutes after baking, followed by storing at room temperature in zip plastic bags.

[0287] The baked doughs will be evaluated on day 1, 7, 14, 21 and 30 for bread samples and on week 2, 4, 6 and 8 for cake samples. Prior to evaluation testing, samples are prepared using a commercial bread-slicing, cutting slices of 12.5 mm thick. End crust slices are discarded, and only central slices are to be used. To achieve AACC standard slice thickness, 2 slices are used per measurement (2* 12.5 mm=25 mm), and 6-10 slices per loaf bread / cake (total 3-5 measurements per data point). Parameters to be evaluated include at least one of the following:a) Texture profile: Hardness and Springinessb) Sensory profile: Softness to touch, Softness to eat, Firmness, Moistness, and Preferability.

[0288] Other relevant parameters, such as loss of resilience over storage, cohesiveness, water mobility, strength, elasticity, stability, stickiness, extensibility, machineability, volume, flavor, crumb structure, crumb softness, blistering, crispiness, resilience both initially and after storage, anti-staling, development time, foldability, flexibility, stackability, flexibility, clumping, flavor retention, hairline cracks, leavening effect, mouthfeel, softness on squeeze, damage during transport, breaking during transport, and sliceability, may be also evaluated.

[0289] Texture analysis will be performed following an adaptation of AACC Method 74.09 (measure of force in compression and springiness or according to AIBCAKE1 / P1 method: Texture Analyzer -The TA. XTplus tester (Stable Micro Systems) and software to capture force-time curve is fitted with a 5kg load cell and 36 mm diameter cylindrical probe (P / 36) before carrying out the test. The probe test compresses the sample until it has compressed 25% of the product height, 2 slices (25mm*25% = 6.25mm). Then measure the maximum force needed to press the probe 7mm into the crumb. It holds at this distance for 30 seconds and then withdraws from the sample and returns to its starting position. Firmness is defined as the force (in grams, kilograms or Newtons) required to compress the product by a pre-set distance e.g. 25 %. A simple way of looking at the springiness property is to record the force after 30 seconds and divide this by the maximum force and then multiply by 100%, according to following formula:F30 / Fmax X 100 = % recovery

[0290] The closer the resulting value is to 100%, the more like a “spring” the product is.

[0291] Based on the products behavior, hardness (or softness), springiness, cohesiveness, gumminess (valid only for semi-solid products), chewiness, fracturability and resilience can be quantified. This study focuses on hardness (firmness) and springiness.

[0292] The following parameters are set:• Test type: Hold until time• Test mode: Measure force in compressionPre test speed: 1.0 mm / sec• Test speed: 1.0 mm / sec• Post test speed: 10.0 mm / sec• Distance: 7.0 mm• Hold time: 32 sec• Trigger time and force: Auto 5g• Data acquisition rate: 200 PPS

[0293] A sensory evaluation after storage will be performed on day 1, 7, 14, 21 and 30 for bread samples and on week 2, 4, 6 and 8 for cake samples for doughs with the alpha-amylase of the present invention and controls / comparator doughs. A larger panel sensory evaluation is optionally performed on day 13. It will be a paired comparison test where a control dough is compared to dough of the present invention.

[0294] Experiments are ongoing showing that the dough comprising the variant polypeptide having alpha-amylase activity of the invention show at least one of the following altered features in comparison to doughs without maltogenic amylase and / or comprising the amylase of SEQ ID NO: 1 and / or comprising a commercial amylase:c) Reduced hardness after storage;d) Improved springiness profile after storage;e) Improved softness to touch;f) Improved softness to eatg) Improved moistness, andh) Improved preferability.SEQUENCE LISTINGSequencesSequence 1: "SEQ ID NO:1"Length Molecule Organism Contains Skipped Type DNA and Sequence RNAfragments686 AA Geobacillus stearothermophilus No No FeaturesFeature Key Feature QualifiersLocationFeature Key Feature QualifiersLocationsource 1..686 mol_type = proteinorganism = Geobacillus stearothermophilusResiduesSSSASVKGDV IYQII IDRFY DGDTTNNNPA KSYGLYDPTK SKWKMYWGGD LEGVRQKLPY 60LKQLGVTT I W LS PVLDNLDT LAGTDNTGYH GYWTRDFKQI EEHFGNWTTF DTLVNDAHQN 120GIKVIVDFVP NHSTPFKAND STFAEGGALY NNGTYMGNYF DDATKGYFHH NGDISNWDDR 180YE WKZIFTE1PAGFSLADLS QENGTIAQYL TDAAVQLVAH GADGLRIDAV KHFNSGFSKS 24QLADlJli Z K.-ZD IFLVGEWYGD DPGTANHLEK VRYAN SGVN VLDFDLNTVI RNVFGTFTQT 300MIDLI IFTU TGNEYKYKEN LI FIDNHD SRFLSVNSNK ANLHQALAFI LTSRGTPSIY 360YGTE Z YI IAGG NDPYNRGM4P AFDTTTTAFK EVSTLAGLRR NNAAIQYGTT TQRWINNDVY 420I YEP I’FFZTDV VLVAINRNTQ S SYS ISGLQT AL NGS AD LSGLLGGN I SVSNGSVASF 480TLAPGAVSVW QYSTSASAPQ IGSVAPNMGI PGNWTIDGK GFGTTQGTVT FGGVTATVKS 540WTSNRIEVYV PNMAAGLTDV KVTAGGVSSN LYSYNILSGT QTSWFTVKS APPTNLGDKI 600YLTGNIPELG NWSTDTSGAV NNAQGPLLAP NYPDWFYVFS VPAGKTIQFK FFIKRADGTI 0QWENGSNHVA TTPTGATGNI TVTWQN 686Sequence 2: " SEQ ID NO:2"Length Molecule Organism Contains Skipped Type DNA and Sequence RNAfragments686 AA unidentified No No FeaturesFeature Key Feature QualifiersLocationsource 1..686 mol-type = proteinorganism = unidentifiedVAR. SEQ 270ResiduesSSSASVKGDV IYQII IDRFY DGDTTNNNPA KSYGLYDPTK SKWKMY GGD LEGVRQKLPY 60LKQLGVTT I LSPVLDNLDT L GTDNTGYH GYWTRDFKQT EEHFGNWTTF DTLVNDAHQN 120GIKVIVDFVP NHSTPFKAND STFAEGGALY NNGTYMGKYF DDATKGYFHH KGDISNWDDR 180YEAZWKNFTD PAGFSLADLS QENGTIAQYL TDAAVQLV H GADGLRIDAV KHF SGFSKS 240LADRLiY K JD IFLVGEWYGD DPGTANHLER VRYA NSGVN VLDFDLNTVI RNVFGTFTQT 300M. DmiP Lu TGNEYKYKEN LITFIDNHD SRFLSVNSNK ANLHQALAFI LTSRGTPSIY 360YGTEZ KDP TJR MMP AFDTTTTAFK EVSTLAGLRR ZLIAAI LYGTT TQRWINNDVY 420VLVAIf® TQ SSYSISGLQT ALPNGSYADY L: L-LL I-.-NZ-Z SVSNGSVASF 480TLAPGAVSW QYSTSASAPQ IGSVAPNMGI PGNWTIDGK Z-F Z-TTZ _-T T FGGVTATVKS 540TSNRIEYW PNMAAGLTDV KVTAGGVSSF LYSYNILSGT.1 FI ’Z. APPTNLGDKI 600^YLTGKIPELG NWSTDTSGAV N AQGPLLAP NYPDWFYVFS VPAGKTIQFK FFIKRADGTI 660QWENGS HVA TTPTGATGNI TVT Q 686

Claims

CLAIMS:

1. A variant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 85 % identity to SEQ ID NO:1 and comprising an amino acid substitution in at least one amino acid position selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and F639.

2. The variant polypeptide according to claim 1, comprising an amino acid substitution in at least two amino acid positions of SEQ ID NO: 1 selected from the group consisting of K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, 1576, V585, V638 and F639.

3. The variant polypeptide according to claim 1 or 2, wherein the amino acid substitution in at least one amino acid position of SEQ ID NO: 1 is selected from the group consisting of K270, L571, V585, E101, M156 and F639, more preferably from the group consisting of K270, L571, V585 and F639.

4. The variant polypeptide according to anyone of claims 1-3, wherein the amino acid substitution in at least one amino acid position of SEQ ID NO: 1 is K270.

5. The variant polypeptide according to anyone of claims 1-4, which comprises the amino acid substitutions in the following positions of SEQ ID NO: 1:a) K270 and L571;b) K270 and V585;c) K270, L571 andV585.

6. The variant polypeptide according to any of the preceding claims, wherein:when the amino acid position K270 is substituted, the amino acid substitution is selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, K270A, K270Q, K270E, K270M, K270P, K270T, K270W and K270Y; preferably the substitution is selected from K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F or K270S, more preferably the substitution is selected from K270R and K270H; even more preferably the substitution is K270R;when the amino acid position T25 is substituted, the amino acid substitution is selected from the group consisting of T25A, T25R, T25N, T25D, T25C, T25Q, T25E, T25G, T25H, T25I, T25L,T25K, T25M, T25F, T25P, T25S, T25W, T25Y, and T25V, preferably from the group consisting of T25G, T25A, T25V, T25L, T25I, and T25P, more preferably the substitution is T25A;when the amino acid position Q99 is substituted, the amino acid substitution is selected from the group consisting of Q99R, Q99A, Q99N, Q99D, Q99C, Q99E, Q99G, Q99H, Q99I, Q99L, Q99K, Q99M, Q99F, Q99P, Q99S, Q99T, Q99W, Q99Y, and Q99V; preferably the substitution is Q99R, Q99K or Q99H, even more preferably the substitution is Q99R;when the amino acid position E101 is substituted, the amino acid substitution is selected from the group consisting ofElOlD, E101A, E101R, E101N, E101C, E101Q, E101G, E101H, E10 II, E101L, E101K, E101M, E101F, E101P, E101S, E101T, E101W, E101Y and E101V, preferably the substitution is selected between E101D and E101E;when the amino acid position A138 is substituted, the amino acid substitution is selected from the group consisting of A138V, A138R, A138N, A138D, A138C, A138Q, A138E, A138G, A138H, A138I, A138L, A138K, A138M, A138F, A138P, A138S, A138T, A138W, and A138Y; preferably the substitution is selected from the group consisting of A138G, A138V, A138L, A138I and A138P; even more preferably the substitution is A138V;when the amino acid position Y 155 is substituted, the amino acid substitution is selected from the group consisting of Y155L, Y155A, Y155R, Y155N, Y155D, Y155C, Y155Q, Y155E, Y155G, Y155H, Y155I, Y155K, Y155M, Y155F, Y155P, Y155S, Y155T, Y155W and Y155V; preferably the substitution is selected from the group consisting ofY155G, Y155A, Y155V, Y155L, Y155I and Y155P; even more preferably the substitution is Y155L;when the amino acid position Ml 56 is substituted, the amino acid substitution is selected from the group consisting of M156I, M156A, M156R, M156N, M156D, M156C, M156Q, M156E, M156G, M156H, M156L, M156K, M156F, M156P, M156S, M156T, M156W, M156Y, and M156V; preferably the substitution is selected from the group consisting of M156G, M156A, M156V, M156L, and M156P; even more preferably the substitution is Ml 561;when the amino acid position W185 is substituted, the amino acid substitution is selected from the group consisting ofW185Y, W185A, W185R, W185N, W185D, W185C, W185Q, W185E, W185G,W185H, W185I, W185L, W185K, W185M, W185F, W185P, W185S, W185T, and W185V; preferably the substitution is selected between W185Y and W185F; more preferably the substitution is W185Y;when the amino acid position A265 is substituted, the amino acid substitution is selected from the group consisting of A265P, A265S, A265R, A265N, A265D, A265C, A265Q, A265E, A265G, A265H, A265I, A265L, A265K, A265M, A265F, A265T, A265W, A265Y, and A265V; preferably the substitution is selected from the group consisting of A265G, A265V, A265L, A265I, A265N, A265Q, A265T, A265C, and A265M; even more preferably the substitution is selected between A265P and A265S;when the amino acid position N309 is substituted, the amino acid substitution is selected from the group consisting of N309T, N309A, N309R, N309D, N309C, N309Q, N309E, N309G, N309H, N309I, N309L, N309K, N309M, N309F, N309P, N309S, N309W, N309Y, and N309V, preferably the substitution is selected from the group consisting of N309Q, N309S, N309T, N309C, and N309M; even more preferably the substitution is N309T;when the amino acid position K318 is substituted, the amino acid substitution is selected from the group consisting of K318E, K318I, K318A, K318R, K318N, K318D, K318C, K318Q, K318G, K318H, K318L, K318M, K318F, K318P, K318S, K318T, K318W, K318Y, and K318V, preferably the substitution is selected from the group consisting of K318E, K318D, K318I, K138G, K138A, K138V, K138L and K138P; even more preferably the substitution is selected between K318E and K318I;when the amino acid position A368 is substituted, the amino acid substitution is selected from the group consisting of A368S, A368R, A368N, A368D, A368C, A368Q, A368E, A368G, A368H, A368I, A368L, A368K, A368M, A368F, A368P, A368T, A368W, A368Y, and A368V; preferably the substitution is selected from the group consisting of A368N, A368Q, A368T, A368C, A368M and A368S; even more preferably the substitution is A368S;when the amino acid position A478 is substituted, the amino acid substitution is selected from the group consisting of A478S, A478R, A478N, A478D, A478C, A478Q, A478E, A478G, A478H, A478I, A478L, A478K, A478M, A478F, A478P, A478T, A478W, A478Y, and A478V, preferably the substitution is selected from the group consisting of A478N, A478Q, A478T, A478C, A478M andA478S; even more preferably the substitution is A478S; when the amino acid position L571 is substituted, the amino acid substitution is selected from the group consisting of L571G, L571S, L571A, L571R, L571N, L571D, L571C, L571Q, L571E, L571H, L571I, L571K, L571M, L571F, L571P, L571T, L571W, L571Y, and L571V; preferably the substitution is selected from the group consisting of L571G, L571A, L571V, L571I, L571P, L571N, L571Q, L571S, L571T, L571C and L571M; even more preferably the substitution is selected between L571G and L571S;when the amino acid position 1576 is substituted, the amino acid substitution is selected from the group consisting of I576L, I576V, I576A, I576R, I576N, I576D, I576C, I576Q, I576E, I576G, I576H, I576K, I576M, I576F, I576P, I576S, I576T, I576W, and I576Y; preferably the substitution is selected from the group consisting of I576G, I576A, I576P I576L or I576V; even more preferably the substitution is selected between I576L and I576V;. when the amino acid position V585 is substituted, the amino acid substitution is selected from the group consisting of V585R, V585A, V585N, V585D, V585C, V585Q, V585E, V585G, V585H, V585I, V585L, V585K, V585M, V585F, V585P, V585S, V585T, V585W, and V585Y; preferably the substitution is selected from the group consisting of V585R, V585K, and V585H; even more preferably the substitution is V585R or V585D;when the amino acid position V638 is substituted, the amino acid substitution is selected from the group consisting of V638D, V638A, V638R, V638N, V638C, V638Q, V638E, V638G, V638H, V638I, V638L, V638K, V638M, V638F, V638P, V638S, V638T, V638W, and V638Y; preferably the substitution is selected between V638D and V638E, more preferably the substitution is V638D;when the amino acid position F639 is substituted, the amino acid substitution is selected from the group consisting of F639V, F639A, F639R, F639N, F639D, F639C, F639Q, F639E, F639G, F639H, F639I, F639L, F639K, F639M, F639P, F639S, F639T, F639W, and F639Y; preferably the substitution is selected from the group consisting of F639V, F639G, F639A, F639L, F639I, F639P; even more preferably the substitution is F639V.

7. The variant polypeptide according to any of the preceding claims, comprising at least one amino acid substitution in SEQ ID NO: 1 selected from the group consisting of K270R, K270V, K270G, K270N, K270D, K270C, K270H, K270I, K270L, K270F, K270S, T25A, Q99R, E101D, A138V,Y155L, M156I, W185Y, A265P, A265S, N309T, K318E, K318I, A368S, A478S, L571G, L571S, I576L, I576V, V585R, V585D, V638D, and F639V; preferably among the group consisting of K270R, E101D, M156I, L571G, L571S, V585R and F639V; more preferably among the group consisting of K270R, E101D, M156I, L571G, L571S, V585D and F639V, even more preferably among the group consisting of K270R, L571G, L571S and V585R, still more preferably among the group consisting of K270R, L571G, L571S and V585D.

8. The variant polypeptide according to anyone of the preceding claims, characterized by the presence of the amino acid substitution K270R, having the amino acid sequence SEQ ID NO:2.

9. The variant polypeptide according to any of the preceding claims, comprising an amino acid substitution in at least two amino acid positions in SEQ ID NO: 1 selected from the pair group consisting of K270+N309, K270+K318, K270+A368, K270+A478, K270+L571, K270+I576, K270+V585, K270+V638, K270+F639, T25+T68, T25+D85, T25+R95, T25+Q99, T25+EI0I, T25+A138, T25+Y155, T25+M156, T25+W185, T25+A265, T25+K270, T25+N309, T25+K318, T25+A368, T25+A478, T25+L571, T25+I576, T25+V585, T25+V638, T25+F639, Q99+EI0I, Q99+A138, Q99+Y155, Q99+M156, Q99+W185, Q99+A265, Q99+K270, Q99+N309, Q99+K318, Q99+A368, Q99+A478, Q99+L571, Q99+I576, Q99+V585, Q99+V638, Q99+F639, E101+A138, E101+Y155, E101+M156, E101+W185, E101+A265, E101+K270, E101+N309, E101+K318, E101+A368, E101+A478, EI0I+L57I, E101+I576, E101+V585, E101+V638, E101+F639, A138+Y155, A138+M156, A138+W185, A138+A265, A138+K270, A138+N309, A138+K318, A138+A368, A138+A478, AI38+L57I, A138+I576, A138+V585, A138+V638, A138+F639, Y155+M156, Y155+W185, Y155+A265, Y155+K270, Y155+N309, Y155+K318, Y155+A368, Y155+A478, YI55+L57I, Y155+I576, Y155+V585, Y155+V638, Y155+F639, M156+W185, M156+A265, M156+K270, M156+N309, M156+K318, M156+A368, M156+A478, MI56+L57I, M156+I576, M156+V585, M156+V638, M156+F639, W185+A265, W185+K270, W185+N309, W185+K318, W185+A368, W185+A478, W185+L57I, W185+I576, W185+V585, W185+V638, W185+F639, A265+K270, A265+N309, A265+K318, A265+A368, A265+A478, A265+L571, A265+I576, A265+V585, A265+V638, A265+F639, N309+K318, N309+A368, N309+A478, N309+L571, N309+I576, N309+V585, N309+V638, N309+F639, K318+A368, K318+A478, K3I8+L57I,K318+I576, K318+V585, K318+V638, K318+F639, A368+A478, A368+L571, A368+I576, A368+V585, A368+V638, A368+F639, A478+L571, A478+I576, A478+V585, A478+V638, A478+F639, L571+I576, L571+V585, L571+V638, L571+F639, I576+V585, I576+A629, I576+V638, I576+F639, V585+V638, V585+F639 and V638+F639; preferably from the group consisting of K270+L571, K270+V585, K270+F639, E101+M156, E101+K270, E101+L571, E101+V585, E101+F639, M156+K270, M156+L571, M156+V585, M156+F639, L571+V585, L571+F639, and V585+F639; even more preferably selected between K270+L571 and K270+V585.

10. The variant polypeptide according to claim 8, comprising an amino acid substitution in at least two amino acid positions of SEQ ID NO: 2 selected from the pair group consisting of T25+T68, T25+D85, T25+R95, T25+Q99, T25+E101, T25+A138, T25+Y155, T25+M156, T25+W185, T25+A265, T25+N309, T25+K318, T25+A368, T25+A478, T25+L571, T25+I576, T25+V585, T25+V638, T25+F639, Q99+E101, Q99+A138, Q99+Y155, Q99+M156, Q99+W185, Q99+A265, Q99+N309, Q99+K318, Q99+A368, Q99+A478, Q99+L571, Q99+I576, Q99+V585, Q99+V638, Q99+F639, E101+A138, E101+Y155, E101+M156, E101+W185, E101+A265, E101+N309, E101+K318, E101+A368, E101+A478, E101+L571, E101+I576, E101+V585, E101+V638, E101+F639, A138+Y155, A138+M156, A138+W185, A138+A265, A138+N309, A138+K318, A138+A368, A138+A478, A138+L571, A138+I576, A138+V585, A138+V638, A138+F639, Y155+M156, Y155+W185, Y155+A265, Y155+N309, Y155+K318, Y155+A368, Y155+A478, Y155+L571, Y155+I576, Y155+V585, Y155+V638, Y155+F639, M156+W185, M156+A265, M156+N309, M156+K318, M156+A368, M156+A478, M156+L571, M156+I576, M156+V585, M156+V638, M156+F639, W185+A265, W185+N309, W185+K318, W185+A368, W185+A478, W185+L571, W185+I576, W185+V585, W185+V638, W185+F639, A265+N309, A265+K318, A265+A368, A265+A478, A265+L571, A265+I576, A265+V585, A265+V638, A265+F639, N309+K318, N309+A368, N309+A478, N309+L571, N309+I576, N309+V585, N309+V638, N309+F639, K318+A368, K318+A478, K318+L571, K318+I576, K318+V585, K318+V638, K318+F639, A368+A478, A368+L571, A368+I576, A368+V585, A368+V638, A368+F639, A478+L571, A478+I576, A478+V585, A478+V638, A478+F639, L571+I576, L571+V585, L571+V638, L571+F639, I576+V585, I576+A629, I576+V638, I576+F639, V585+V638, V585+F639and V638+F639; preferably from the group consisting of E101+M156, E101+L571, E101+V585, E101+F639, M156+L571, M156+V585, M156+F639, L571+V585, L571+F639 and V585+F639.

11. The variant polypeptide according to any of the preceding claims, which comprises at least one further amino acid substitution in combination with at least one amino acid substitutions in the position K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, I576, V585, V638 and / or F639 of SEQ ID NO: 1, said further amino acid substitution occurring in a position selected from the group consisting of Y273, T68, D85, R95, I125, P130, F136, S141, T142, N151, A163, R180, E182, D198, L199, Q201, H232, F233, S235, Y258, G259, D260, D285, M367, F427, Q449, S488, S493, T494, T528, V534, M553 and A629.

12. The variant polypeptide according to any of the preceding claims, which comprises at least one further amino acid substitution in combination with at least one amino acid substitutions in the position T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, I576, V585, V638 and / or F639 of SEQ ID NO:2, said further amino acid substitution occurring in a position selected from the group consisting of Y273, T68, D85, R95, I125, P130, F136, S141, T142, N151, A163, R180, E182, D198, L199, Q201, H232, F233, S235, Y258, G259, D260, D285, M367, F427, Q449, S488, S493, T494, T528, V534, M553 and A629.

13. The variant polypeptide according to anyone of claim 11 or 12, wherein said at least one further amino acid substitution is selected from the group consisting ofY273F, T68A, D85G, R95S, R95H, I125C, I125Y, P130T, F136Y, S141A, T142S, T142G, N151D, A163S, R180G, R180D, R180Y, R180C, E182N, E182C, D198S, D198L, L199I, Q201V, Q201D, Q201C, H232D, F233M, S235E, S235A, S235F, S235M, Y258F, Y258A, Y258V, G259T, G259M, G259A, G259Y, G259V, G259S, G259H, D260G, D260S, D285R, M367L, F427G, F427A, Q449N, S488G, S493T, T494A, T528S, V534T, M553A, A629S and A629C.

14. The variant polypeptide according to anyone of claims 11 or 13, wherein said at least one further amino acid substitution in combination with at least one amino acid substitution occurring in position K270, T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, I576, V585, V638 and / or F639 of SEQ ID NO: 1 is selected from the group consisting ofK270R+Y273F, K270R+M367L, K270R+Q449N, K270R+M553A, M156I+R180G, M156I+F233M, M156I+D260G, M156I+D260S, M156I+Y273F, M156I+M367L, M156I+Q449N, M156I+M553A, R180G+L571S, R180G+V585R, R180G+V585D, R180G+F639V, F233M+L571S, F233M+V585R, F233M+V585D, F233M+F639V, D260G+L571S, D260G+V585R, D260G+V585D, D260G+F639V, D260S+L571S, D260S+V585R, D260S+V585D, D260S+F639V, Y273F+L571S, Y273F+V585R, Y273F+V585D, Y273F+F639V, M367L+L571S, M367L+V585R, M367L+V585D, M367L+F639V, Q449N+L571S, Q449N+V585R, Q449N+V585D, Q449N+F639V, M553A+L571S, M553A+V585R, M553A+V585D and M553A+F639V; preferably from the group consisting of D260S+K270R, R180G+K270R, M553A+K270R and K270R+Y273F15. The variant polypeptide according to anyone of claim 12 or 13, wherein said at least one further amino acid substitution in combination with at least one amino acid substitution occurring in position T25, Q99, E101, A138, Y155, M156, W185, A265, N309, K318, A368, A478, L571, I576, V585,V638 and / or F639 of SEQ ID NO:2 is selected from the group consisting of M156I+R180G, M156I+F233M, M156I+D260G, M156I+D260S, M156I+Y273F, M156I+M367L, M156I+Q449N, M156I+M553A, R180G+L571S, R180G+V585R, R180G+V585D, R180G+F639V, F233M+L571S, F233M+V585R, F233M+V585D, F233M+F639V, D260G+L571S, D260G+V585R, D260G+V585D, D260G+F639V, D260S+L571S, D260S+V585R, D260S+V585D, D260S+F639V, Y273F+L571S, Y273F+V585R, Y273F+V585D, Y273F+F639V, M367L+L571S, M367L+V585R, M367L+V585D, M367L+F639V, Q449N+L571S, Q449N+V585R, Q449N+V585D, Q449N+F639V, M553A+L571S, M553A+V585R, M553A+V585D and M553A+F639V.

16. The variant polypeptide according to any of the preceding claims, wherein the amino acid sequence has at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.

17. The variant polypeptide according to any of the preceding claims, wherein the amino acid sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2.

18. The variant polypeptide according to any of the preceding claims, wherein the variant shows a maltogenic alpha-amylase activity, wherein the variant is capable of hydrolysing (1→4)-α-d-glyco-sidic linkages in a glucose polymer comprising glucose molecules linked by alpha- 1,4-glycosidic bonds into malto-oligosaccharides.

19. The variant polypeptide according to claim 18, wherein the glucose polymer is a starch selected from the group consisting of soluble starch, resistant starch, and damaged starch.

20. The variant polypeptide according to claim 18, wherein the malto-oligosaccharide is maltose.

21. The variant polypeptide according to any of the preceding claims, wherein the variant exhibits at least one altered feature in comparison with SEQ ID NO: 1, wherein the altered feature is (A), (B), or any combination thereof; wherein:(A) is an increase in enzymatic activity of at least 1.1 -fold relative to the alpha-amylase of SEQ ID NO: 1 under standard test conditions; and(B) is an increased sucrose tolerance, wherein the sucrose tolerance is determined as enzymatic activity in the presence of sucrose, wherein the enzymatic activity in the presence of sucrose is at least 1.1-fold higher than the enzymatic activity in the presence of sucrose of the alphaamylase of SEQ ID NO: 1 under standard test conditions.

22. The variant polypeptide according to any of the preceding claims, wherein the dough or baked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

23. A composition comprising the variant polypeptide according to any one of claims 1 to 22 together with one or more additional components.

24. The composition according to claim 23, wherein the composition is a dough conditioner composition comprising the variant polypeptide according to any one of claims 1 to 22, and wherein said one or more additional components is selected from the group consisting of diluents, additional enzymes,emulsifiers, oxidizing agents, reducing agents, hydrocolloids, surfactants, dough conditioner agents, flavor enhancers, preservatives, and nutritional supplements.

25. The composition according to any one of claims 23-24, wherein the composition is a pre-mix composition comprising the variant polypeptide according to any one of claims 1 to 22 and flour or a mixture of flour.

26. Use of a variant polypeptide according to any one of claims 1 to 22 or of a composition according to any one of claims 23 to 25, for the preparation of a dough and / or a baked product.

27. The use according to claim 26, wherein the dough or baked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

28. A dough or a baked product comprising a variant polypeptide according to any one of claims 1 to 22, or a composition according to any one of claims 23 to 25.

29. The dough or baked product according to claim 28, wherein the dough or baked product comprises sugar and / or sucrose in an amount ranging from at least 5 wt% to 40 wt% based on total flour weight, or from at least 10 wt% to 40 wt% sugar and / or sucrose based on weight of total ingredients.

30. A process for the production of a dough or baked product comprising adding a variant polypeptide according to any one of claims 1 to 22, or a composition according to any one of claims 23 to 25 to the dough or baked product according to claim 28 or 29.

Images