WO2021032881A1

WO2021032881A1 - Amylase variants

Info

Publication number: WO2021032881A1
Application number: PCT/EP2020/073514
Authority: WO
Inventors: Stefan Jenewein; Zachary David MILES; Priya ANAND
Original assignee: Basf Se
Priority date: 2019-08-22
Filing date: 2020-08-21
Publication date: 2021-02-25
Also published as: JP2022545465A; US20220267748A1; EP4017974A1; BR112022000573A2; CN114364795A; MX2022002182A

Abstract

Genetically engineered enzymes having amylase enzyme activity, compositions comprising the enzymes, and methods of making and using the enzymes. The genetically engineered amylase enzymes are useful in many different applications such as laundry detergents, dish washing detergents, and cleaning products for homes, industry, vehicle care, baking, animal feed, pulp and paper processing, starch processing, brewing, and ethanol production.

Description

AMYLASE VARIANTS

TECHNICAL FIELD

In the present invention new amylase enzymes are provided. More specifically, genetically engineered amylase enzymes, compositions comprising the enzymes, and methods of using the enzymes or compositions comprising the enzymes. The genetically engineered amylase enzymes are useful in many different applications such as laundry detergents, dish washing detergents, and cleaning products for homes, industry, vehicle care, baking, animal feed, pulp and paper processing, starch processing, and ethanol production. Amylases have been employed in the removal of starch stains and have been added to various compositions such as cleaning products. A lot of these applications require the use of the amylases being either stable at elevated temperatures or within a denaturing condition. Thus, the need exists for genetically engineered amylase enzymes with improved properties, in particular with improved stability and improved performance.

SUMMARY OF THE INVENTION

The present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha- amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44.

Preferably, the polypeptide having alpha-amylase activity consists of an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44.

Preferably, the A and B domain of the polypeptide having alpha-amylase activity has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the A and B domain having the amino acid sequence of SEQ ID NO: 42.

Preferably, the C domain of the polypeptide having alpha-amylase activity has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the C domain having the amino acid sequence of SEQ ID NO: 44.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution, deletion, and/or insertion at one or more positions.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence

TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDI TGNQTNT VTINXD GXGQFXVXXGSXSI YXQX, wherein X can be any ammo acid. Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has one or more amino acid residues selected from the group consisting of 402R,H; 419S,G,D;

420V, I; 422 A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W;

473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39. Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably the amylase of the present invention comprises amino acid residues 430M and/or 4301, preferably the substitution resulting in 430M and/or 4541, preferably the substitution being selected from the group consisting of I430M and M454I, according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 432, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO: 39, preferably one or more substitutions selected from the group consisting of M9L, El 30V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S and E190P according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises:

(a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37;

(b) an amino acid sequence encoded by a polynucleotide having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38,

(c) an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with the complement of

(I) a coding sequence of SEQ ID NO: 54, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or

(II) a polynucleotide shown in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; or

(d) a fragment of (a), (b), or (c) having amylase activity.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an increase in expression, activity, thermostability, stability, performance in laundry, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, or any combination thereof compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40, preferably, the amylase has an increase in thermostability compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40.

The present invention also refers to an isolated, a synthetic, or a recombinant nucleic acid comprising:

(a) a nucleic acid sequence having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO : 6, SEQ ID NO : 8, SEQ ID NO: 10, SEQ ID NO : 12, SEQ ID NO : 14, SEQ ID NO : 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity;

(b) a nucleic acid sequence encoding a polypeptide having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO: 37, wherein the polypeptide has amylase activity or any polypeptide described herein having amylase activity;

(c) a polynucleotide that hybridizes under high stringency conditions with the complement of (I) a coding sequence of SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or (h) a polynucleotide shown in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38;

(d) a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having amylase activity; or

(e) a nucleic acid sequence fully complementary to any of (a) to (d).

The present invention also refers to a nucleic acid construct comprising the polynucleotide as described herein.

The present invention also refers to an expression vector comprising the polynucleotide or the nucleic acid construct as described herein.

The present invention also refers to a host cell comprising the polynucleotide as described herein, the nucleic acid construct as described herein, or the expression vector as described herein.

The present invention also refers to a composition comprising the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity as described herein.

Preferably, the composition further comprising at least one second enzyme selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a mannanase, a pectinase, xylanase, and a nuclease.

The present invention also refers to a method of making the isolated, synthetic, or recombinant polypeptide having alpha-amylase as described herein, comprising: providing a nucleic acid sequence encoding the polypeptide, transforming the nucleic acid sequence into an expression host, cultivating the expression host to produce the polypeptide, and optionally purifying the polypeptide.

The present invention also refers to a method of preparing a dough or a baked product prepared from the dough, the method comprising adding the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity as described herein to the dough and baking it.

The present invention also refers to a method of use of the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity as described herein, for processing starch, for cleaning or washing textiles, hard surfaces, or dishes, for making ethanol, for processing pulp or paper, or for feeding an animal.

The present invention also refers to a method of making an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising the step of making a hybrid from at least two different amylases, wherein the hybrid comprises an A and B domain and a C domain and wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44.

The present invention also refers to a method of use of a C domain of a first amylase said C domain having an amino acid sequence which has at least 75% identity to the amino acid sequence of SEQ ID NO: 44 for improving one or more properties selected from the group consisting of stability, pH profile, expression, activity, thermostability, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, performance in laundry, processing starch, cleaning textiles, cleaning hard surfaces, cleaning dishes, making ethanol, processing pulp or paper, and feeding an animal of a second alpha amylase having an A and B domain with at least 75 % identity to the amino acid sequence of SEQ ID NO: 42 said use comprising replacing the C domain of the second alpha-amylase with the C domain of the first alpha-amylase.

DETAILED DESCRIPTION OF THE INVENTION An enzyme is a biological molecule (polypeptide) comprising a sequence of amino acid residues, wherein the enzyme can catalyze a reaction. Hence, enzymes are catalytically active proteins or polypeptides. Enzyme names are determined based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Enzymes are defined by an EC (Enzyme Commission) number, recommended name, alternative names (if any), catalytic activity, and other factors. Enzymes herein may be identified by polypeptide sequences (also called amino acid sequences herein). The polypeptide sequence specifies the three-dimensional structure including the “active site” of an enzyme which in turn determines the catalytic activity of the same. Polypeptide sequences may be identified by a SEQ ID NO.

Enzymes are obtained from or derived from many different sources including: plants; animals; bacteria, archaea, fungi, yeast, environmental samples containing DNA that encodes an enzyme, or enzymes can be synthetic generated in a laboratory. For example, bacterial sources of enzymes include enzymes derived from Bacillus, Streptomyces, E. coli and Pseudomonas, fungal sources of enzymes include enzymes derived from Aspergillus, Fusarium, Thermomyces and Trichoderma yeast sources of enzymes include enzymes derived from Pichia, and Saccharomyces .

The World Intellectual Property Office (WIPO) Standard ST.25 (1998) provides that the amino acid residues should be represented in the sequence listing using the following three-letter symbols with the first letter as a capital. The table below provides an overview of the amino acid identifiers as well as the corresponding DNA codons that encode the amino acid using the standard genetic standard. The DNA codons that encode amino acid residues can be different depending organism that is used and slightly different tables for translation of the genetic code may apply. A compilation of such non-standard code translation tables is maintained at the NCBI.

A “parent” polypeptide amino acid sequence is the starting sequence for introduction of mutations (e.g. by introducing one or more (fragments) amino acid substitutions, insertions, deletions, or a combination thereof) to the sequence, resulting in “variants” of the parent polypeptide amino acid sequences. A parent includes: A wild-type polypeptide amino acid sequence or synthetically generated polypeptide amino acid sequence that is used as starting sequence for introduction of (further) changes.

A “variant polypeptide” refers to an enzyme that differs from its parent in its amino acid sequence. The differences between the parent polypeptide and variant polypeptide can be one single amino acid residue, or more than one amino acid residue. The more than one amino acid residue and be consecutive amino acid residues or non-consecutive amino acid residues. The consecutive amino acid residues can be four consecutive amino acid residues; five consecutive amino acid residues; eight consecutive amino acid residues; nine consecutive amino acid residues; eleven consecutive amino acid residues; thirteen consecutive amino acid residues; or fourteen consecutive amino acid residues. While the definition below describes variants in the context of amino acid changes, nucleic acids may be similarly modified, e.g. by substitutions.

A “mature polypeptide” means an enzyme in its final form including any post-translational modifications, glycosylation, phosphorylation, truncation, N-terminal modifications, C-terminal modifications, signal sequence deletion. A mature polypeptide can vary depending upon the expression system, vector, promoter, and/or production process.

A "synthetic" or “artificial” compound is produced by in vitro chemical or enzymatic synthesis.

The term “non-naturally occurring” refers to a (poly)nucleotide, amino acid, (poly)peptide, enzyme, protein, cell, organism, or other material that is not present in its original naturally occurring environment or source. Preferably, the amylase of the present invention is a non- naturally occurring amylase.

Variant polynucleotide and variant polypeptide sequences may be defined by their sequence identity when compared to a parent sequence. Sequence identity usually is provided as “% sequence identity” or “% identity”. For calculation of sequence identities, in a first step a sequence alignment is produced. According to this invention, a pairwise global alignment is produced, meaning that two sequences are aligned over their complete length, which is usually produced by using a mathematical approach, called alignment algorithm.

According to the invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453). Preferably, the program “NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (polynucleotides: gap open=10.0, gap extend=0.5 and matrix=EDNAFLEL; polypeptides: gap open=10.0, gap extend=0.5 and matrix=EBLO SUM62) .

After aligning two sequences, in a second step, an identity value is determined from the alignment produced.

For this purpose, the %-identity is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of the present invention over its complete length multiplied with 100: %-identity = (identical residues / length of the alignment region which is showing the respective sequence of the present invention over its complete length) * 100.

For calculating the percent identity of two nucleic acid sequences the same applies as for the calculation of percent identity of two amino acid sequences with some specifications. For nucleic acid sequences encoding for a protein the pairwise alignment shall be made over the complete length of the coding region of the sequence of this invention from start to stop codon excluding introns. Introns present in the other sequence, to which the sequence of this invention is compared, may also be removed for the pairwise alignment. Percent identity is then calculated by %-identity = (identical residues / length of the alignment region which is showing the sequence of the invention from start to stop codon excluding introns over their complete length)

* 100. After aligning two sequences, in a second step, an identity value is determined from the alignment produced.

Moreover, the preferred alignment program for nucleic acid sequences implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453) is “NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EDNAFULL).

Sequences, having identical or similar regions with a sequence of this invention, and which shall be compared with a sequence of this invention to determine % identity, can easily be identified by various ways that are within the skill in the art, for instance, using publicly available computer methods and programs such as BLAST, BLAST-2, available for example at NCBI.

Variant polypeptides may be defined by their sequence similarity when compared to a parent sequence. Sequence similarity usually is provided as “% sequence similarity” or “%- similarity”. % sequence similarity takes into account that defined sets of amino acids share similar properties, e.g. by their size, by their hydrophobicity, by their charge, or by other characteristics. Herein, the exchange of one amino acid with a similar amino acid may be called “conservative mutation”. Similar amino acids according to the invention are defined as follows, which shall also apply for determination of %-similarity according to this invention, which is also in accordance with the BLOSUM62 matrix as for example used by program “NEEDLE”, which is one of the most used amino acids similarity matrix for database searching and sequence alignments: Amino acid A is similar to amino acids S Amino acid D is similar to amino acids E; N Amino acid E is similar to amino acids D; K; Q Amino acid F is similar to amino acids W; Y Amino acid H is similar to amino acids N; Y Amino acid I is similar to amino acids L; M; V Amino acid K is similar to amino acids E; Q; R Amino acid L is similar to amino acids I; M; V Amino acid M is similar to amino acids I; L; V Amino acid N is similar to amino acids D; H; S Amino acid Q is similar to amino acids E; K; R Amino acid R is similar to amino acids K; Q Amino acid S is similar to amino acids A; N; T Amino acid T is similar to amino acids S Amino acid V is similar to amino acids I; L; M Amino acid W is similar to amino acids F; Y Amino acid Y is similar to amino acids F; H; W

Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme. In one embodiment, such mutations are not pertaining the functional domains of an enzyme. In one embodiment, conservative mutations are not pertaining the catalytic centers of an enzyme.

For calculation of sequence similarity, in a first step a sequence alignment is produced as described above. After aligning two sequences, in a second step, a similarity value is determined from the alignment produced.

For this purpose, the %-similarity is calculated by dividing the number of identical residues plus the number of similar residues by the length of the alignment region which is showing the sequence of the invention over its complete length multiplied with 100: %-similarity = [(identical residues + similar residues) / length of the alignment region which is showing the sequence of the invention over its complete length] * 100. The invention relates to a polypeptide having amylase activity comprising an amino acid sequence that is at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to any one of the full length amino acid sequence of : SEQ ID NO:54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

The invention further relates to a polynucleotide encoding a variant polypeptide of the invention. The terms "polynucleotide(s)", "nucleic acid sequence(s)", "nucleotide sequence(s)", “nucleic acid(s)”, “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length. A “gene” is a DNA segment carrying a certain genetic information.

A “parent” polynucleotide acid sequence is the starting sequence for introduction of mutations to the sequence, resulting in “variants” of said parent polynucleotide sequence. A “variant polynucleotide” refers to a polynucleotide that encodes an enzyme and the variant polynucleotide differs from its parent polynucleotide in its nucleic acid sequence.

The polynucleotide of the invention in one aspect has a nucleic acid sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical when compared to any one of the full length polynucleotide sequence of SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38.

Preferably, the polynucleotide is a codon-optimized polynucleotide for improving expression in a specific host cell.

“Substitutions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the substituted amino acid. A specific amino acid residue may be substituted with any of the 19 amino acid residues different from the original one. For example, the substitution of histidine at position 120 with alanine is designated as “His 120 Ala” or “H120A”. Alternative substitutions at an amino acid position are indicated as follows “His 120 Ala, Leu” or “H120A,L”. It is understood herein that instead of the indicated specific substitutions alternative substitutions using conservative amino acid alternatives can be used.

Amino acid deletions are described by providing the original amino acid of the parent enzyme followed by the number of the position within the amino acid sequence, followed by *. Accordingly, the deletion of glycine at position 150 is designated as “Glyl50*” or G150*”. Alternatively, deletions are indicated by e.g. “deletion of D183 and G184”.

Amino acid insertions are described by providing the original amino acid of the parent enzyme followed by the number of the position within the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, an insertion at position 180 of lysine next to glycine is designated as “Glyl80GlyLys” or “G180GK”. When more than one amino acid residue is inserted, such as e.g. a Lys and Ala after Glyl80 this may be indicated as: Glyl80GlyLysAla or G195GKA.

In cases where a substitution and an insertion occur at the same position, this may be indicated as S99SD+S99A or in short S99AD.

The one or more amino acid substitution of the variant polypeptides can be one or more conservative amino acid substitution. A “conservative amino acid substitution” or “related amino acid” means replacement of one amino acid residue in an amino acid sequence with a different amino acid residue having a similar property at the same position compared to the parent amino acid sequence. Some examples of a conservative amino acid substitution include but are not limited to replacing a positively charged amino acid residue with a different positively charged amino acid residue; replacing a polar amino acid residue with a different polar amino acid residue; replacing a non-polar amino acid residue with a different non-polar amino acid residue, replacing a basic amino acid residue with a different basic amino acid residue, or replacing an aromatic amino acid residue with a different aromatic amino acid residue.

“Enzymatic activity” means at least one catalytic effect exerted by an enzyme.

Enzymatic activity is expressed as units per milligram of enzyme (specific activity) or molecules of substrate transformed per minute per molecule of enzyme (molecular activity). Enzymatic activity can be specified by the enzymes actual function, e.g. proteases exerting proteolytic activity by catalyzing hydrolytic cleavage of peptide bonds, lipases exerting lipolytic activity by hydrolytic cleavage of ester bonds, amylases activity involves (endo)hydrolysis of glucosidic linkages in polysaccharides, etc.

Enzymatic activity may change during storage or operational use of the enzyme. The term “enzyme stability” relates to the retention of enzymatic activity as a function of time during storage or operation. The term “storage” herein means to indicate the fact of products or compositions or formulations being stored from the time of being manufactured to the point in time of being used in final application. Retention of enzymatic activity as a function of time during storage may be called “storage stability” herein.

To determine and quantify changes in catalytic activity of enzymes stored or used under certain conditions over time, the “initial enzymatic activity” is measured under defined conditions at time zero (100%) and at a certain point in time later (x%). By comparison of the values measured, a potential loss of enzymatic activity can be determined in its extent. The extent of enzymatic activity loss determines an enzymes stability or non-stability.

Parameters influencing the enzymatic activity of an enzyme and/or storage stability and/or operational stability are for example pH, temperature, chelators, and presence of oxidative substances.

A variant polypeptide may be active over a broad pH at any single point within the range from about pH 4.0 to about pH 12.0. The variant polypeptides enzyme may be active over a range of pH 5.0 to pH 11.0, pH 6.0 to pH 10.0, and pH 7.0 to pH 9.0. In another embodiment, the variant polypeptides enzyme may be active over a pH 7.1 to pH 8.9, pH 7.2 to pH 8.8, pH 7.3 to pH 8.7, pH 7.4 to pH 8.6, pH 7.5 to pH 8.5. The variant polypeptides may be active at pH 4.0, pH 4.1, pH

4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH

5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH

6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH

7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6 pH 8.7, pH 8.8 pH 8.9, pH

9.0, pH 9.1, pH 9.2, pH 9.3, pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH 9.9, pH 10.0, pH 10.1, pH

10.2, pH 10.3, pH 10.4, pH 10.5, pH 10.6, pH 10.7, pH 10.8, pH 10.9, pH 11.0, pH 11.1, pH 11.2, pH 11.3, pH 11.4, pH 11.5, pH 11.6, pH 11.7, pH 11.8, pH 11.9, pH 12.0, pH 12.1, pH 12.2, pH

12.3, pH 12.4, and pH 12.5, pH 12.6, pH 12.7, pH 12.8, pH 12.9, or higher. A “pH stability”, refers to the ability of an enzyme to exert enzymatic activity at a specific pH range.

The variant polypeptides may be active over a broad temperature, wherein the temperature is any point in the range from about 10 °C to about 95°C. The variant polypeptides may be active at a temperature range from 10 ° C to 55 ° C, 10 ° C to 50 ° C, 10 ° C to 45 ° C, 10 ° C to 40 ° C, 10 ° C to 35 ° C, 10 ° C to 30 ° C, or 10 ° C to 25° C The variant polypeptides may be active at a temperature range from 20 ° C to 55 ° C, 20 ° C to 50 ° C, 20 ° C to 45 ° C, 20 ° C to 40 ° C, 20 ° C to 35 ° C, 20 ° C to 30 ° C, or 20 ° C to 25° C. The variant polypeptides are active at a temperature of at least 10 ° C, 11 ° C, 12 ° C, 13 ° C, 14 ° C, 15 ° C, 16 ° C, 17 ° C, 18 ° C, 19 ° C, 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 ° C, 63 ° C, 64 ° C, 65 ° C, 66 ° C, 67 ° C, 68 ° C, 69 ° C, 70 ° C, 71 ° C, 72 ° C, 73 ° C, 74 ° C, 75 ° C, 76 ° C, 77 ° C, 78 ° C, 79 ° C, 80 ° C, 81 ° C, 82 ° C, 83 ° C, 84 ° C, 85 ° C, 86 ° C, 87 ° C, 88 ° C, 89 ° C, 90 ° C, 91 ° C, 92 ° C, 93 ° C, 94 ° C, 95 ° C, or higher temperatures.

The terms “thermal stability” and “thermostability” refer to the ability of a protein to exert catalytic activity at a specific temperature range. Enzymes thermostability may be characterized by what is known as the T value (also called half-life, see above). The T indicates the temperature at which 50% residual enzymatic activity is still present after thermal inactivation for a certain time when compared with a reference sample which has not undergone thermal treatment.

In one embodiment, the variant polypeptides improve the thermostability compared to the parent molecule. In another embodiment the variant polypeptides improve the thermostability by 3 C, 4 C, 5 C, 6 C, 7 C, 8 C, 9 C, 10 C, 11 C, 12 C, 13 C, 14 C, 15 C, 16 C, 17 C, 18 C, 19 C, 20 C, or more degrees C when compared to the parent polypeptide. In another embodiment, the thermostability increase is measured at a temperature between 65 C and 100° C. The thermostability increase can be measured at 65 C, 66 C, 67 C, 68 C, 69 C, 70 C, 71 C, 72 C, 73 C, 74 C, 75 C, 76 C, 77 C, 78 C, 79 C, 80 C, 81 C, 82 C, 83 C, 84 C, 85 C, 86 C, 87 C, 88 C, 89 C, 90 C, 91 C, 92 C, 93 C, 94 C, 95 C, 96 C, 97 C, 98 C, 99 C, and/or 100 C. In another embodiment, the thermostability increase is measured at a temperature of 70° C. In another embodiment, the thermostability increase is measured at a temperature of 80° C. In another embodiment, the thermostability increase is measured a temperature of 90° C. In one embodiment, the thermostability is improved at 70°C, at 80°C, or at 90°C, preferably at 70°C. In another embodiment, the thermostability is improved in a temperature range between 65 °C and 90°C, preferably between 70 °C and 85 °C, preferably between 70 °C and 80 °C.

In one embodiment, the variant polypeptide is a fragment of the full-length amino acid sequence and the fragment has amylase activity.

A "Fragment", or “subsequence” as used herein are a portion of a polynucleotide or an amino acid sequence.

The term “functional fragment” refers to any nucleic acid or amino acid sequence which comprises merely a part of the full-length amino acid sequence, respectively, but still has the same or similar activity and/or function. Preferably, the functional fragment is at least 75% identical, at least 76% identical, at least 77% identical, at least 78% identical, at least 79% identical, at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5 %, at least 99%, or at least 99.5% identical to the full length amino acid sequence original sequence. The functional fragment comprises contiguous nucleic acids or amino acids compared to the original nucleic acid or original amino acid sequence, respectively.

A-, B- and C-domains:

The structure of alpha-amylases comprises three distinct domains A, B and C, see, e.g., Machius et al., 1995, J. Mol. Bio/ 246: 545-559. The term "domain" means a region of a polypeptide that in itself forms a distinct and independent substructure of the whole molecule. Alpha-amylases consist of a beta/alpha-8 barrel harboring the active site residues, which is denoted the A- domain, a rather long loop between the beta-sheet and alpha-helix 3, which is denoted the B- domain (together; "A and B domain"), and a C-domain and in some cases also an additional carbohydrate binding domain (e.g., WO 2005/001064; Machius etal, supra).

The domains of an alpha-amylase can be determined by structure analysis such as using crystallographic techniques. An alternative method for determining the domains of an alpha- amylase is by sequence alignment of the amino acid sequence of the alpha-amylase with another alpha-amylase for which the domains have been determined. The sequence that aligns with, e.g., the C-domain sequence in the alpha-amylase for which the C-domain has been determined can be considered the C-domain for the given alpha-amylase.

A and B domain:

The term "A and B domain" as used herein means these two domains taken as one unit, whereas the C domain is another unit of the alpha-amylases. Thus, the amino acid sequence of the "A and B domain" is understood as one consecutive sequence or one part of a sequence of an alpha- amylase comprising an "A and B domain" and other, additional domains (such as the C domain). Thus, the term "the A and B domain has at least 75% sequence identity to SEQ ID NO: 42" means that the amino acid sequence that form the A and B domain has at least 75% sequence identity to SEQ ID NO: 42. As used herein, the "A and B domain" of an alpha-amylase corresponds to amino acids 1-399 of SEQ ID NO: 39.

AB domain donor: the term AB domain donor as used herein means the alpha-amylase from which the A and B domain is obtained. Thus, for the A and B domain having the amino acid sequence of SEQ ID NO: 42, the AB domain donor is the alpha-amylase of SEQ ID NO: 39.

C domain:

As used herein, the "C domain" of an alpha-amylase corresponds to amino acids 400-485 of for example SEQ ID NO: 39. Thus, the C domain of an alpha amylase may be found by alignment of said alpha amylase with the alpha amylase of SEQ ID NO: 39 The part of said alpha amylase that aligns with amino acids 400-485 of SEQ ID NO: 39 is according to the present invention "the C domain" of the alpha amylase. Thus, for instance, the C domain of the alpha amylase having the amino acid sequence of SEQ ID NO: 40 is made up of amino acids 401-486 disclosed here as SEQ ID NO: 44.

Carbohydrate binding domain or carbohydrate binding module (CBM):

The amylases comprised of catalytic modules (A, B and C domain) may further comprise one or more non-catalytic CBMs (carbohydrate-binding modules, also called carbohydrate binding domain or specifically for amylases starch binding domains). CBMs can improve the association of the enzyme with the substrate. CBMs are attached to the C-domain.

Alpha-amylases of the present invention comprise three domains; A, B and C domains. Preferably, the amylase of the present invention does not comprise a carbohydrate binding domain. Preferably, alpha-amylases of the present invention consists only of the three domains being A, B and C domain.

The inventors of the present invention have surprisingly found, that a polypeptide which is a hybrid of the A and B domain from a first alpha amylase (the " AB domain donor") of SEQ ID NO: 39 or variants thereof and the C domain from a second alpha amylase (the "C domain donor") of SEQ ID NO: 40 or variants thereof has improved properties, compared to the alpha- amylase of the AB domain donor (SEQ ID NO: 39 or variants thereof) and the alpha-amylase of the C domain donor (SEQ ID NO: 40 or variants thereof) and/or even the alpha-amylase of SEQ ID NO: 41, which is the alpha-amylase of SEQ ID NO: 39 having a stability improving mutation, i.e., a deletion at amino acid position 182 and 183 according to the numbering of SEQ ID NO:

39.

The A and B domain of the alpha-amylase having the amino acid sequence of SEQ ID NO: 39 were determined to correspond to amino acids 1-399. This sequence is also disclosed as SEQ ID NO: 42 herein. The C domain of the amino acid sequence of SEQ ID NO: 39 was determined to correspond to amino acids 400-485 (disclosed herein as SEQ ID NO: 43). The C domain of the alpha-amylase having the amino acid sequence of SEQ ID NO: 40 was determined to correspond to amino acids 401-486 of SEQ ID NO: 40 and is also disclosed as SEQ ID NO: 44 herein. Thus, in one embodiment of the present invention, the polypeptide having alpha-amylase activity is a hybrid of amino acids 1-399 of SEQ ID NO: 39 or variants thereof and amino acids 401-486 of SEQ ID NO: 40 or variants thereof.

AB domain donors:

In one embodiment, the A and B domain is obtained from the alpha-amylase comprising the amino acid sequence of SEQ ID NO: 39 which A and B domain is also disclosed herein as SEQ ID NO: 42. In one embodiment of the present invention, the amino acid sequence forming the A and B domain has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42.

Other suitable AB domain donors are alpha-amylases closely related to the alpha-amylase of SEQ ID NO: 39. Preferably, AB domain donors are alpha-amylases having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase of SEQ ID NO: 39. Preferably, AB domain donors are alpha-amylases having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase of SEQ ID NO: 41.

Alternatively, AB domain donors are alpha-amylases having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase of SEQ ID NO: 40.

C domain donors:

The most preferred C domain donor is the alpha-amylase disclosed as SEQ ID NO: 40 from which the C domain is determined to correspond to amino acids 401-486 which is also disclosed as SEQ ID NO: 44 herein. Accordingly, the invention relates in the most preferred embodiments to the above disclosed A and B domains fused with the C domain disclosed as SEQ ID NO: 44 or a C domain having at least 75% sequence identity hereto. In another embodiment the invention relates to alpha-amylases comprising the above disclosed A and B domains fused with a C domain having a sequence which is at least 80% identical to the sequence of SEQ ID NO: 44. In another embodiment the invention relates to alpha-amylases comprising the above disclosed A and B domains fused with a C domain having a sequence which is at least 85% identical to the sequence of SEQ ID NO: 44. In another embodiment the invention relates to alpha-amylases comprising the above disclosed A and B domains fused with a C domain having a sequence which is at least 90% identical to the sequence of SEQ ID NO: 44. In another embodiment the invention relates to alpha-amylases comprising the above disclosed A and B domains fused with a C domain having a sequence which is at least 95% identical to the sequence of SEQ ID NO:

44. In another embodiment the invention relates to alpha-amylases comprising the above disclosed A and B domains fused with a C domain having a sequence which is at least 97% identical to the sequence of SEQ ID NO: 44. In another embodiment the invention relates to alpha-amylases comprising the above disclosed A and B domains fused with a C domain having a sequence which is at least 98% identical to the sequence of SEQ ID NO: 44. In another embodiment the invention relates to alpha-amylases comprising the above disclosed A and B domains fused with a C domain having a sequence which is at least 99% identical to the sequence of SEQ ID NO: 44. In another embodiment the invention relates to alpha-amylases comprising the above disclosed A and B domains fused with a C domain having a sequence which is 100% identical to the sequence of SEQ ID NO: 44.

Suitable C domains which are at least 75% identical to the C domain of SEQ ID NO: 44 are the two C domains disclosed as SEQ ID NOs 46 and 48 herein. Respective hybrid amylases hereof are shown in SEQ ID NO: 49 and 50.

Preferably, C domain donors are alpha-amylases having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha- amylase of SEQ ID NO: 40.

Alternatively, C domain donors are alpha-amylases having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha- amylase of SEQ ID NO: 39.

Thus, in an alternative embodiment, in case AB domain donors are alpha-amylases having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase of SEQ ID NO: 40, then C domain donors are alpha- amylases having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase of SEQ ID NO: 39.

Hybrids:

The present invention refers to amylases (i.e., polypeptides having amylase activity), which can be considered as hybrids between the amylase shown in SEQ ID NO: 39 and the amylase shown in SEQ ID NO: 40 and variants thereof having amylase activity. In one embodiment, the invention refers to hybrid amylases comprising an A and B domain from the amylase shown in SEQ ID NO. 39 and the C domain of the amylase shown in SEQ ID NO: 40 and variants thereof having amylase activity.

In an alternative embodiment, the invention refers to hybrid amylases comprising an A and B domain from the amylase shown in SEQ ID NO. 40 and the C domain of the amylase shown in SEQ ID NO: 39 and variants thereof having amylase activity. Thus, the polypeptide of the present invention may be described as a hybrid or a fusion polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper etal, 1993, EMBO J. 12: 2575-2583; Dawson et ah, 1994, Science 266: 776-779). The polypeptide according to the invention may alternatively be produced by synthetic gene construction by means known to the skilled person. Thus, it is not necessary that the A and B domain on the one hand and the C domain on the other hand of the claimed polypeptides are derived from different alpha amylases and fused together. They may e.g. also be synthetically produced for instance by introducing the respective amino acid substitutions in a parent amylase sequence and thereby creating a variant amylase, which equals to a hybrid sequence. Thus, the amylase of the present invention can be obtained by a method of making an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising the step of making a hybrid from at least two different amylases, wherein the hybrid comprises an A and B domain and a C domain and wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44. Alternatively, the amylase of the present invention can be obtained by a method comprising the step of modifying a parent amylase (preferably as shown in SEQ ID NO: 39 or SEQ ID NO: 40 or variants thereof) and introducing into this parent amylase amino acid substitutions at certain position, preferably at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,

69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,

115, 116, 117, 118, 119, 120, 121, 122, or 123 positions, preferably 1-123, preferably, at 1-29 positions, more preferably, at 1-20 or even more preferably, 1-10 positions.

Preferably, the parent amylase is an amylase shown in SEQ ID NO: 39 and within the C domain of SEQ ID NO: 39 one or more amino acid substitutions are introduced, preferably at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 positions, preferably, at 1-29 positions, more preferably, at 1-10 positions, to convert the amino acid sequence at these positions to SEQ ID NO: 40.

Preferably, the alpha-amylase has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase shown in SEQ ID NO: 39 and within the C domain of SEQ ID NO: 39 the amino acid residue at one or more of the amino acid positions selected from the group consisting of the positions 400, 402, 408, 409,

410, 418, 419, 420, 422, 423, 429, 430, 437, 441, 444, 446, 449, 452, 454, 458, 459, 460, 466,

471, 473, 475, 482, 484, and 485 according to the numbering of SEQ ID NO: 39 is exchanged, preferably to an amino acid residue present in SEQ ID NO: 40.

Preferably, the alpha-amylase has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase shown in SEQ ID NO: 39 and within the C domain of SEQ ID NO: 39 the amino acid sequence comprises one or more substitutions selected from the group consisting of K400T, N402R, H408P, N409D, M410V, N418D, T419G, A420V, P422A, N423D, I429L, M430I, N437S, Y441E, R444K, K446N, Q449E, R452Y, I454M, R458Q, S459T, G460N, A466K, N471Q, S473H, N475S, W482Y, N484Q, and N485Q according to the numbering of SEQ ID NO: 39.

Alternatively, the parent amylase is an amylase shown in SEQ ID NO: 40 and within the AB domain of SEQ ID NO: 40 one or more amino acid substitutions are introduced, preferably at 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,

82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,

106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or 123 positions, preferably 1-123, more preferably 1-100, even more preferably 1-50 positions, to convert the amino acid sequence at these positions to SEQ ID NO: 39.

Preferably, the alpha-amylase has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase shown in SEQ ID NO: 40 and within the A and B domain of SEQ ID NO: 40 the amino acid residue at one or more of the amino acid positions selected from the group consisting of the positions 1, 2, 3, 4, 5,

9, 17, 25, 28, 29, 32, 35, 36, 41, 48, 51, 52, 82, 83, 86, 87, 89, 90, 93, 94, 95, 96, 98, 113, 116, 118, 123, 124, 125, 129, 136, 138, 142, 144, 150, 158, 165, 169, 170, 172, 174, 183, 186, 192,

193, 206, 208, 212, 214, 217, 218, 222, 225, 227, 229, 235, 242, 243, 244, 245, 246, 250, 251,

255, 256, 260, 263, 267, 269, 273, 274, 275, 276, 280, 282, 284, 286, 291, 297, 298, 299, 302,

303, 304, 311, 313, 318, 320, 323, 324, 328, 330, 337, 338, 339, 343, 345, 346, 355, 356, 360,

361, 374, 375, 376, 377, 378, 379, 382, 384, 391, 394, 395, and 396 according to the numbering of SEQ ID NO: 39 is exchanged, preferably to an amino acid residue present in SEQ ID NO: 39. Preferably, the alpha-amylase has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase shown in SEQ ID NO: 40 and within the A and B domain of SEQ ID NO: 40 the amino acid sequence comprises one or more substitutions selected from the group consisting of the positions A1HH, A2H, T3N, I4G, N5T, L9M, A17L, K25N, H28R, T29S, G32S, A35K, Q36D, S41A, Y48W, T51A, T52S, K82R, A83N, K86Q, S87A, I89V, E90T, H93K, K94S, Q95N, N96G, N98Q, Y113A, T116W, T118R, D123N, R124P, N125S, I129Q, E136T, N138E, G142K, N144D, D150N, K158R, T165V, E169Q, G170S, K172Q, L173LQ, I183D, A186G, S192D, S193T, L206I, F208M, D212E, A214V, M217L, K218R, T222V, A225T, E227T, N229G, L235I, D242K, H243Y, E244S, Y245F, L246T, V250L, N251T, Q255N, Q256T, E260N, T263A, Y267F, Q269K, Q273G, T274A, L275I, N276E, A280S, V282T, Y284W, Q286H, A291V, F297L, H298Y, Y299N, K302R, G303S, N304G, N311Q, L313F, M318V, N320R, A323T, L324H, L328F, E330D, G337E, Q338E, S339A, V343F, S345E, P346E, F355L, I356T, A360D, E361Q, TSGN374I, S375P, S376T, Y377H, E378G, I379V, L382M, D384S, M391E, K394Q, N395K, and F396Y according to the numbering of SEQ ID NO: 39.

In one embodiment, the hybrid amylase comprises the A and B domain from an amylase having an amino acid sequence with at least 75% sequence identity to an amylase shown in SEQ ID NO. 39 and the C domain from an amylase having an amino acid sequence with at least 75% sequence identity to the amylase shown in SEQ ID NO: 40.

In one embodiment of the present invention, the amino acid sequence forming the A and B domain has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

42, and the amino acid sequence forming the C domain has at least 75% identity to SEQ ID NO: 44.

The alpha-amylases may be produced by substituting the C domain or a portion thereof of an alpha-amylase with the C domain or a portion thereof of another alpha-amylase. When producing a hybrid alpha-amylase, no amino acids should be deleted or inserted in the linker region, wherein the linker region is understood herein as amino acid positions 380-420 according to the numbering of SEQ ID NO: 39. Preferably, amino acid positions 380-420 according to the numbering of SEQ ID NO: 39 of the amylase comprises amino acid residues as present in either SEQ ID NO: 39 and / or SEQ ID NO: 40. Preferably, amino acid positions 390-410 according to the numbering of SEQ ID NO: 39 of the amylase comprises amino acid residues as present in either SEQ ID NO: 39 and / or SEQ ID NO: 40. Preferably, amino acid positions 395-405 according to the numbering of SEQ ID NO: 39 of the amylase comprises amino acid residues as present in either SEQ ID NO: 39 and / or SEQ ID NO: 40. Preferably, the amylase comprises at amino acid positions 380-399 (according to the numbering of SEQ ID NO: 39) the amino acid residues of SEQ ID NO: 39. Preferably, the amylase comprises at amino acid positions 400-420 (according to the numbering of SEQ ID NO: 39) the amino acid residues of SEQ ID NO: 40. Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution, deletion, and/or insertion at one or more positions.

420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39. In a further embodiment, the invention relates to a variant of the polypeptides disclosed above. The amylase of the invention may comprise additional substitutions, deletions, and/or insertions at one or more positions. The polypeptides may be mutated (substitution, deletion, and/or an insertion) in the A and B domain only, or in the C domain only or in both the A and B domain and the C domain. The polypeptide may be mutated (substitution, deletion, and/or an insertion) outside the A and B domain and the C domain in case additional residues are present, e.g., a carbohydrate binding domain. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine sequence, an antigenic epitope or a binding domain.

In one embodiment, the variant of the amylase of SEQ ID NO:54, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 comprising a substitution at one or more positions and having amylase activity comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions, preferably, at 1-20 positions, more preferably, at 1-10 positions, preferably conservative substitutions.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for alpha-amylase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton etal, 1996, J. Bio/. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos etal, 1992, Science 255: 306-312; Smith etal, 1992, J. Mol. Bio/. 224: 899- 904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide. Essential amino acids in the sequence of amino acids of SEQ ID NO: 39 are located at positions D236, E266 and D333, which are the catalytic residues. These should preferable not be mutated. Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad Sei. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman etal, 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et ah, 1986, Gene 46: 145; Ner etal., 1988, DNA 7: 127). Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et ah, 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

Preferred mutations are deletions of at one or more, preferably at least 2, amino acids positions selected from 181, 182, 183 and 184, such as amino acids 181+182, or 182+183, or 181+183 or 181+184 of SEQ ID NO: 39. Hereby the molecule is considerably stabilized. In a preferred embodiment of the invention, the amino acids corresponding to 182 and 183 in SEQ ID NO: 39 are deleted. In one embodiment, the hybrid amylase of the present invention comprises the A and B domain of SEQ ID NO: 39, or a variant as disclosed herein, and the C domain from an alpha amylase of SEQ ID NO: 40, or a variant as disclosed herein, and further comprises a deletion of the amino acids corresponding to 182 and 183 in SEQ ID NO: 39. Such variant is disclosed for example as SEQ ID NO: 54 herein.

In other embodiment of the invention, in the hybrid amylase comprises amino acid substitutions within the interface of the domain C and the A and B domain in order to avoiding steric clashes. In one embodiment preferred substitutions are substitutions within the C domain of the donor amylase (such as SEQ ID NO: 44) into the amino acid of the C domain to be replaced (such as SEQ ID NO: 43) at the equal position. Equal positions can be defined by aligning both sequences. Preferred positions for these mutations are analyzed by inspection of structural model. Preferred, but not restricted to, are substitutions at one or more of the following amino acid positions within the donor C domain: 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (numbering according to SEQ ID NO: 39). Preferably, the polypeptide having amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39.

In another embodiment preferred substitutions are substitutions within the A and B domain of the C domain acceptor amylase (such as SEQ ID NO: 42) into the amino acid of the A and B domain of the C domain donor amylase (such as SEQ ID NO: 51) present at the equal position. Equal positions can be defined by aligning both sequences. Preferred positions for these mutations are analyzed by inspection of structural model. Preferred, but not restricted to, are substitutions at one or more of the following amino acid positions within the A and B domain of the C domain acceptor amylase; preferably within the A and B domain of SEQ ID NO: 39: 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (numbering according to SEQ ID NO: 39). Preferably, the polypeptide having amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40.

An example for the adaptation of the interface within a hybrid amylase consisting out of an A and B domain as given in SED ID NO: 42 and the C domain as given in SEQ ID NO: 44 resulting in a hybrid amylase as given in SEQ ID NO: 52 are the mutations I430M and M454I resulting in an amylase given as SEQ ID NO: 53 or in SEQ ID NO: 54 (numbering according to SEQ ID NO: 39). Thus, preferably, the amylase of the present invention comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably, the amylase of the present invention comprises amino acid residues 430M and/or 4301, preferably the substitution resulting in 430M and/or 4541, preferably the substitution being selected from the group consisting of I430M and M454I, according to the numbering of SEQ ID NO: 39.

The polypeptide having amylase activity of the present invention may further comprise a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181 , 186, and 190, preferably, at one or more positions selected from the group consisting of 9, 179, 186, 195, and 206, more preferably, one or more positions selected from the group consisting of 179, 195, and 206, according to the numbering of SEQ ID NO: 39, preferably one or more substitutions selected from the group consisting of M9L, El 30V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S, andE190P, preferably, one ormore substitutions selected from the group consisting of M9L, K179L, G186E/N/Q/S, N195F, and I206L, more preferably, one or more substitutions selected from the group consisting of K179L, G186E, N 195F, and I206L, according to the numbering of SEQ ID NO: 39.1n a further embodiment, the invention also relates to polynucleotides encoding the amylases of the present invention. In a further embodiment, the invention also relates to polypeptides which are encoded by a polynucleotide that hybridizes high stringency conditions with (i) the mature polypeptide coding sequence as described herein or (ii) the full-length complement of (i). The term "hybridisation" as defined herein is a process wherein substantially complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to a carrier, including, but not limited to a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. This formation or melting of hybrids is dependent on various parameters, including but not limited thereto the temperature. An increase in temperature favours melting, while a decrease in temperature favours hybridisation. However, this hybrid forming process is not following an applied change in temperature in a linear fashion: the hybridisation process is dynamic, and already formed nucleotide pairs are supporting the pairing of adjacent nucleotides as well. So, with good approximation, hybridisation is a yes-or-no process, and there is a temperature, which basically defines the border between hybridisation and no hybridisation. This temperature is the melting temperature (Tm). Tm is the temperature in degrees Celsius, at which 50% of all molecules of a given nucleotide sequence are hybridised into a double strand, and 50% are present as single strands.

The melting temperature (Tm) is dependent from the physical properties of the analysed nucleic acid sequence and hence can indicate the relationship between two distinct sequences. However, the melting temperature (Tm) is also influenced by various other parameters, which are not directly related with the sequences, and the applied conditions of the hybridization experiment must be taken into account. For example, an increase of salts (e.g. monovalent cations) is resulting in a higher Tm.

Tm for a given hybridisation condition can be determined by doing a physical hybridisation experiment, but Tm can also be estimated in silico for a given pair of DNA sequences. In this embodiment, the equation of Meinkoth and Wahl (Anal. Biochem., 138:267-284, 1984) is used for stretches having a length of 50 or more bases: Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500/L.

M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA stretch, % form is the percentage of formamide in the hybridisation solution, and L is the length of the hybrid in base pairs. The equation is for salt ranges of 0.01 to 0.4 M and % GC in ranges of 30% to 75%.

While above Tm is the temperature for a perfectly matched probe, Tm is reduced by about 1°C for each 1% of mismatching (Bonner et al., J. Mol. Biol. 81: 123-135, 1973): Tm = [ 81.5°C + 16.6(log M) + 0.41 (%GC) - 0.61 (%formamide) - 500/L ] - %non-identity.

This equation is useful for probes having 35 or more nucleotides and is widely referenced in scientific method literature (e.g. in: “Recombinant DNA Principles and Methodologies”, James Greene, Chapter “Biochemistry of Nucleic acids”, Paul S. Miller, page 55; 1998, CRC Press), in many patent applications (e.g. in: US 7026149), and also in data sheets of commercial companies (e.g. “Equations for Calculating Tm” from www.genomics.agilent.com).

Other formulas for Tm calculations, which are less preferred in this embodiment, might be only used for the indicated cases:

For DNA-RNA hybrids (Casey, J. and Davidson, N. (1977) Nucleic Acids Res. ,4: 1539):

Tm = 79.8°C +18.5 (log M) + 0.58 (% GC) + 11.8 (%GC * % GC) -0.5 (% form) - 820/L. For RNA-RNA hybrids (Bodkin, D.K. and Knudson, D.L. (1985) J. Virol. Methods, 10: 45):

Tm = 79.8°C +18.5 (log M) + 0.58 (% GC) + 11.8 (%GC * %GC) -0.35 (% form) - 820/L.

For oligonucleotide probes of less than 20 bases (Wallace, R.B., et al. (1979) Nucleic Acid Res.

6: 3535): Tm = 2 x n(A+T) + 4 x n(G+C), with n being the number of respective bases in the probe forming a hybrid. For oligonucleotide probes of 20-35 nucleotides, a modified Wallace calculation could be applied: Tm = 22 + 1.46 n(A+T) + 2.92 n(G+C), with n being the number of respective bases in the probe forming a hybrid.

For other oligonucleotides, the nearest-neighbour model for melting temperature calculation should be used, together with appropriate thermodynamic data:

Tm = (å(AHd)+AHi) / ( å(ASd)+ASi+ASself + Rxln(cT/b) ) + 16.61og[ Na +] - 273.15 (Breslauer, K.J., Frank, R., Blocker, H., Marky, L.A. 1986 Predicting DNA duplex stability from the base sequence. Proc. Natl Acad. Sci. USA 833746-3750; Alejandro Panjkovich, Francisco Melo, 2005. Comparison of different melting temperature calculation methods for short DNA sequences. Bioinformatics, 21 (6): 711-722) where:

Tm is the melting temperature in degrees Celsius; å(AHd) and å(ASd) are sums of enthalpy and entropy (correspondingly), calculated over all internal nearest-neighbor doublets;

ASself is the entropic penalty for self-complementary sequences;

AHi and ASi are the sums of initiation enthalpies and entropies, respectively;

R is the gas constant (fixed at 1.987 cal/K mol); cT is the total strand concentration in molar units; constant b adopts the value of 4 for non-self-complementary sequences or equal to 1 for duplexes of self-complementary strands or for duplexes when one of the strands is in significant excess. The thermodynamic calculations assume that the annealing occurs in a buffered solution at pH near 7.0 and that a two-state transition occurs.

Thermodynamic values for the calculation can be obtained from Table 1 in (Alejandro Panjkovich, Francisco Melo, 2005. Comparison of different melting temperature calculation methods for short DNA sequences. Bioinformatics, 21 (6): 711-722), or from the original research papers (Breslauer, K.J., Frank, R., Blocker, H., Marky, L.A. 1986 Predicting DNA duplex stability from the base sequence. Proc. Natl Acad. Sci. USA 833746-3750; SantaLucia, T, Jr, Allawi, H.T., Seneviratne, P.A. 1996 Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 353555-3562; Sugimoto, N., Nakano, S., Yoneyama, M., Honda, K. 1996 Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res. 244501-4505). For an in silico estimation of Tm according to this embodiment, first a set of bioinformatic sequence alignments between the two sequences are generated. Such alignments can be generated by various tools known to a person skilled in the art, like programs “Blast” (NCBI), “Water” (EMBOSS) or “Matcher” (EMBOSS), which are producing local alignments, or “Needle” (EMBOSS), which is producing global alignments. Those tools should be applied with their default parameter setting, but also with some parameter variations. For example, program “MATCHER” can be applied with various parameter for gapopen/gapextend (like 14/4; 14/2; 14/5; 14/8; 14/10; 20/2; 20/5; 20/8; 20/10; 30/2; 30/5; 30/8; 30/10; 40/2; 40/5; 40/8; 40/10; 10/2; 10/5; 10/8; 10/10; 8/2; 8/5; 8/8; 8/10; 6/2; 6/5; 6/8; 6/10) and program “WATER” can be applied with various parameter for gapopen/gapextend (like 10/0,5; 10/1; 10/2; 10/3; 10/4; 10/6; 15/1; 15/2; 15/3; 15/4; 15/6; 20/1; 20/2; 20/3; 20/4; 20/6; 30/1; 30/2; 30/3; 30/4; 30/6; 45/1; 45/2; 45/3; 45/4; 45/6; 60/1; 60/2; 60/3; 60/4; 60/6), and also these programs shall be applied by using both nucleotide sequences as given, but also with one of the sequences in its reverse complement form. For example, BlastN (NCBI) can be applied with an increased e-value cut-off (e.g. e+1 or even e+10) to also identify very short alignments, especially in data bases of small sizes. Important is that local alignments are considered, since hybridisation may not necessarily occur over the complete length of the two sequences, but may be best at distinct regions, which then are determining the actual melting temperature. Therefore, from all created alignments, the alignment length, the alignment %GC content (in a more accurate manner, the %GC content of the bases which are matching within the alignment), and the alignment identity has to be determined. Then the predicted melting temperature (Tm) for each alignment has to be calculated. The highest calculated Tm is used to predict the actual melting temperature.

The term "hybridisation over the complete sequence of the invention" as defined herein means that for sequences longer than 300 bases when the sequence of the invention is fragmented into pieces of about 300 to 500 bases length, every fragment must hybridise. For example, a DNA can be fragmented into pieces by using one or a combination of restriction enzymes. A bioinformatic in silico calculation of Tm is then performed by the same procedure as described above, just done for every fragment. The physical hybridisation of individual fragments can be analysed by sta8ndard Southern analysis, or comparable methods, which are known to a person skilled in the art. The term "stringency" as defined herein is describing the ease by which hybrid formation between two nucleotide sequences can take place. Conditions of a “higher stringency” require more bases of one sequence to be paired with the other sequence (the melting temperature Tm is lowered in conditions of “higher stringency”), conditions of “lower stringency” allow some more bases to be unpaired. Hence the degree of relationship between two sequences can be estimated by the actual stringency conditions at which they are still able to form hybrids. An increase in stringency can be achieved by keeping the experimental hybridisation temperature constant and lowering the salts concentrations, or by keeping the salts constant and increasing the experimental hybridisation temperature, or a combination of these parameter. Also an increase of formamide will increase the stringency. The skilled artisan is aware of additional parameters which may be altered during hybridisation and which will either maintain or change the stringency conditions (Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).

A typical hybridisation experiment is done by an initial hybridisation step, which is followed by one to several washing steps. The solutions used for these steps may contain additional components, which are preventing the degradation of the analyzed sequences and/or prevent unspecific background binding of the probe, like EDTA, SDS, fragmented sperm DNA or similar reagents, which are known to a person skilled in the art (Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3^rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).

A typical probe for a hybridisation experiment is generated by the random-primed-labelling method, which was initially developed by Feinberg and Vogelstein (Anal. Biochem., 132 (1), 6- 13 (1983); Anal. Biochem., 137 (1), 266-7 (1984) and is based on the hybridisation of a mixture of all possible hexanucleotides to the DNA to be labelled. The labelled probe product will actually be a collection of fragments of variable length, typically ranging in sizes of 100 - 1000 nucleotides in length, with the highest fragment concentration typically around 200 to 400 bp.

The actual size range of the probe fragments, which are finally used as probes for the hybridisation experiment, can also be influenced by the used labelling method parameter, subsequent purification of the generated probe (e.g. agarose gel), and the size of the used template DNA which is used for labelling (large templates can e.g. be restriction digested using a 4 bp cutter, e.g. Haelll, prior labeling).

For the present invention, the sequence described herein is analysed by a hybridisation experiment, in which the probe is generated from the other sequence, and this probe is generated by a standard random-primed-labelling method. For the present invention, the probe is consisting of a set of labelled oligonucleotides having sizes of about 200 - 400 nucleotides. A hybridisation between the sequence of this invention and the other sequence means, that hybridisation of the probe occurs over the complete sequence of this invention, as defined above. The hybridisation experiment is done by achieving the highest stringency by the stringency of the final wash step. The final wash step has stringency conditions comparable to the stringency conditions of at least Wash condition 1: 1.06 x SSC, 0.1 % SDS, 0 % formamide at 50°C, in another embodiment of at least Wash condition 2: 1.06 x SSC, 0.1 % SDS, 0 % formamide at 55°C, in another embodiment of at least Wash condition 3: 1.06 x SSC, 0.1 % SDS, 0 % formamide at 60°C, in another embodiment of at least Wash condition 4: 1.06 x SSC, 0.1 % SDS, 0 % formamide at 65°C, in another embodiment of at least Wash condition 5: 0.52 x SSC, 0.1 % SDS, 0 % formamide at 65°C, in another embodiment of at least Wash condition 6: 0.25 x SSC, 0.1 % SDS, 0 % formamide at 65°C, in another embodiment of at least Wash condition 7: 0.12 x SSC, 0.1 %

SDS, 0 % formamide at 65°C, in another embodiment of at least Wash condition 8: 0.07 x SSC, 0.1 % SDS, 0 % formamide at 65°C.

A “low stringent wash” has stringency conditions comparable to the stringency conditions of at least Wash condition 1, but not more stringent than Wash condition 3, wherein the wash conditions are as described above.

A “high stringent wash” has stringency conditions comparable to the stringency conditions of at least Wash condition 4, in another embodiment of at least Wash condition 5, in another embodiment of at least Wash condition 6, in another embodiment of at least Wash condition 7, in another embodiment of at least Wash condition 8, wherein the wash conditions are as described above.

The amylase of the present invention displays improved properties. Preferably, the improved property is relative to the property of an amylase shown in SEQ ID NO: 39 and/or SEQ ID NO: 40. It is preferred that the wash performance is improved at 15°C.In one embodiment the property that is improved is detergent stability. In another embodiment the property that is improved is specific activity. In another embodiment the property that is improved is thermal stability. In another embodiment the property that is improved is pH-dependent stability. In another embodiment the property that is improved is oxidative stability. In another embodiment the property that is improved is the reduction of Ca2+ dependency. In yet another embodiment the property that is improved is wash performance at low temperature. In one embodiment of the invention the polypeptides have an improved wash performance at low temperatures, such as at 40°C or below 40°C, or at or below 30°C, or at or below 25°C or at or below 20°C or at or below 15°C, or at or below 10°C. In a preferred embodiment, the property that is improved is thermal stability

Preferred polypeptides having amylase activity (amylases):

Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

44.

Preferably, the polypeptide having alpha-amylase activity consists of an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence

TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDI TGNQTNT VTINXD GXGQFXVXXGSXSI YXQX, wherein X can be any ammo acid. Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 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%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is 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%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence

TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDI TGNQTNTVTINXDGXGQFXVXXGSXSIYXQX, wherein X can be any ammo acid. Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has one or more amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39, also preferred, selected from the group consisting of 435R, 437A, 441D, and 485K, according to the numbering of SEQ ID NO: 39. Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or all amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S;

476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably the amylase comprises amino acid residues 430M and/or 4301, preferably the substitution is selected from the group consisting of I430M and M454I, preferably I430M and M454I, according to the numbering of SEQ ID NO: 39.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably the amylase comprises amino acid residues 430M and/or 4301, preferably the substitution selected from the group consisting of I430M and M454I, preferably I430M and M454I, according to the numbering of SEQ ID NO: 39, wherein the polypeptide having alpha- amylase comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably the substitution selected from the group consisting of 1430M and M454I, preferably I430M and M454I, according to the numbering of SEQ ID NO: 39, wherein the polypeptide having alpha-amylase comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39) and wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence

TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDI TGNQTNT VTINXD GXGQFXVXXGSXSI YXQX, wherein X can be any ammo acid, preferably, wherein the polypeptide has one or more amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39.

Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably the amylase comprises amino acid residues 430M and/or 4301, preferably the substitution selected from the group consisting of I430M and M454I, preferably I430M and M454I, according to the numbering of SEQ ID NO: 39, wherein the polypeptide having alpha-amylase comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 52, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably the amylase comprises amino acid residues 430M and/or 4301, preferably the substitution resulting in 430M and/or 4541, preferably the substitution being selected from the group consisting of I430M and M454I, preferably I430M and M454I, according to the numbering of SEQ ID NO: 39, wherein the polypeptide having alpha-amylase comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53, wherein the polypeptide having alpha-amylase comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39. Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13, or all amino acid residues of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all amino acid residues of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion of amino acids corresponding to positions 182 and 183. Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181 , 186, and 190, preferably, at one or more positions selected from the group consisting of 9, 179, 186, 195, and 206, more preferably, one or more positions selected from the group consisting of 179, 195, and 206, according to the numbering of SEQ ID NO: 39, preferably one or more substitutions selected from the group consisting of M9L, El 30V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S, andE190P, preferably, one ormore substitutions selected from the group consisting of M9L, K179L, G186E/N/Q/S, N195F, and I206L, more preferably, one or more substitutions selected from the group consisting of K179L, G186E, N195F, and I206L, according to the numbering of SEQ ID NO: 39.

Also disclosed herein is an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions, preferably at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all amino acid residue positions, selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO: 39, preferably one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S, and E190P according to the numbering of SEQ ID NO: 39.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all amino acid residue positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO: 39, preferably at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all amino acid substitutions selected from the group consisting of M9L, El 30V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S, and E190P according to the numbering of SEQ ID NO: 39.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an increase in expression, activity, thermostability, stability, performance in laundry, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, or any combination thereof compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40, preferably, the amylase has an increase in thermostability compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an increase in thermostability compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40.

In one embodiment, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises:

(a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, preferably, SEQ ID NO:54, SEQ ID NO:5, SEQ ID NO: 11, SEQ ID NO: 17, or SEQ ID NO:27;

(b) an amino acid sequence encoded by a polynucleotide having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, preferably, SEQ ID NO:55, SEQ ID NO:6, SEQ ID NO: 12, SEQ ID NO: 18, or SEQ ID NO:29,

(I) a coding sequence of SEQ ID NO: 54, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, preferably, SEQ ID NO:54, SEQ ID NO:5, SEQ ID NO:ll, SEQ ID NO:17, or SEQ ID NO: 27; or

(II) a polynucleotide shown in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, preferably, SEQ ID NO:55, SEQ ID NO:6, SEQ ID NO: 12, SEQ ID NO: 18, or SEQ ID NO:29; or

(d) a fragment of (a), (b), or (c) having amylase activity.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence

TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDI TGNQTNT VTINXD GXGQFXVXXGSXSI YXQX, wherein X can be any ammo acid. Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDI TGNQTNT VTINXD GXGQFXVXXGSXSI YXQX, wherein X can be any ammo acid, preferably, wherein the polypeptide has one or more amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDI

TGNQTNTVTINXDGXGQFXVXXGSXSIYXQX, wherein X can be any ammo acid.

Preferably, the amylase of the present invention comprises at least one amino acid substitution as described herein and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to the amino acid sequence of: SEQ ID

NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,

SEQ ID NO:35, or SEQ ID NO:37, preferably, SEQ ID NO:54, SEQ ID NO:5, SEQ ID NO: 11, SEQ ID NO: 17, or SEQ ID NO:27.

Preferably, the amylase of the present invention comprises at least one amino acid substitution as described herein and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to the amino acid sequence of: SEQ ID NO: 54, preferably at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to the amino acid sequence of: SEQ ID NO: 54. Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID N0:11, SEQ ID N0:13, SEQ ID N0:15, SEQ ID NO: 17, SEQ ID N0:19, SEQ ID N0:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID N0:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has one or more amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K, preferably, selected from the group consisting of 435R, 437A, 441D, and 485K, according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:39, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has one or more amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K, according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has one or more amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39, preferably, selected from the group consisting of 435R, 437S,A, 441E,D, and 485Q,K, according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or all amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V;

483V; and 485Q according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has amino acid residues 402R,H; 419S,G,D; 420V, I; 422, A, V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID

NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably the amylase comprises amino acid residues 430M and/or 4301, preferably the substitution resulting in 430M and/or 4541, preferably the substitution being selected from the group consisting of I430M and M454I, according to the numbering of SEQ ID NO: 39. Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454, preferably the amylase comprises amino acid residues 430M and/or 4301, preferably the substitution resulting in 430M and/or 4541, preferably the substitution being selected from the group consisting of 1430M and M454I, according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at position 430 and 454, preferably the substitution resulting in 430M and/or 4541, preferably the substitution being selected from the group consisting of 1430M and M454I, according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at position 430 and 454, preferably the substitution resulting in 430M and/or 4541, preferably the substitution being selected from the group consisting of 1430M and M454I, according to the numbering of SEQ ID NO: 39, wherein the polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13, or all amino acid residues of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13, or all amino acid residues of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all amino acid residues of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39). Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 182 and 183, preferably a deletion of both amino acids corresponding to positions 182 and 183 (according to the numbering of SEQ ID NO: 39).

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions

181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and

182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 182 and 183, preferably a deletion of both amino acids corresponding to positions 182 and 183 (according to the numbering of SEQ ID NO: 39). Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of both amino acids corresponding to positions 182 and 183 (according to the numbering of SEQ ID NO: 39). Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO: 39, preferably one or more substitutions selected from the group consisting of M9L, El 30V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S, and E190P according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190, preferably, at one or more positions selected from the group consisting of 9, 179, 186, 195, and 206, more preferably, one or more positions selected from the group consisting of 179, 195, and 206, according to the numbering of SEQ ID NO: 39, preferably one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S, and E190P, preferably, one or more substitutions selected from the group consisting of M9L, K179L, G186E/N/Q/S, N195F, and I206L, more preferably, one or more substitutions selected from the group consisting of K179L, G186E, N195F, and I206L, according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all amino acid residue positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO: 39, preferably at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all amino acid substitutions selected from the group consisting of M9L, El 30V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S, and E190P according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all amino acid residue positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO: 39, preferably at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all amino acid substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S, and E190P according to the numbering of SEQ ID NO: 39.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an increase in expression, activity, thermostability, stability, performance in laundry, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, or any combination thereof compared to the amylase shown in SEQ ID NO:

39 or SEQ ID NO: 40, preferably, the amylase has an increase in thermostability compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an increase in expression, activity, thermostability, stability, performance in laundry, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, or any combination thereof compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40, preferably, the amylase has an increase in thermostability compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40.

Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an increase in thermostability compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40.

Preferably, the present invention refers to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

44, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54.

(a) a nucleic acid sequence having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO: 38, wherein the nucleic acid encodes a polypeptide having amylase activity;

(b) a nucleic acid sequence encoding a polypeptide having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least,

96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID N0:5, SEQ ID N0:7, SEQ ID N0:9, SEQ ID N0:11, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID N0:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity or any polypeptide described herein having amylase activity, preferably a polypeptide having alpha- amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44;

(c) a polynucleotide that hybridizes under high stringency conditions with the complement of (I) a coding sequence of SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or (h) a polynucleotide shown in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38;

(e) a nucleic acid sequence fully complementary to any of (a) to (d).

Preferably, the isolated, a synthetic, or a recombinant nucleic acid comprises:

(a) a nucleic acid sequence having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO: 38, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a nucleic acid sequence encoding a polypeptide having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least,

96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity;

(c) a fragment of (a), or (b), wherein the fragment encodes a polypeptide having amylase activity; or

(d) a nucleic acid sequence fully complementary to any of (a) to (d).

Further preferred herein is an isolated, a synthetic, or a recombinant nucleic acid comprising (a) a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity;

(b) a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to SEQ ID NO:54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity;

(c) a fragment of (a) or (b), wherein the fragment encodes a polypeptide having amylase activity; or

(d) a nucleic acid sequence fully complementary to any of (a) to (c).

Further preferred herein is an isolated, a synthetic, or a recombinant nucleic acid comprising (a) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 55, wherein the nucleic acid encodes a polypeptide having amylase activity;

(b) a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to SEQ ID NO: 54, wherein the polypeptide has amylase activity;

(c) a fragment of (a) or (b), wherein the fragment encodes a polypeptide having amylase activity; or (d) a nucleic acid sequence fully complementary to any of (a) to (c).

Preferably, the isolated, a synthetic, or a recombinant nucleic acid comprises:

96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity.

Preferably, the isolated, a synthetic, or a recombinant nucleic acid comprises:

(a) a nucleic acid sequence having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55, wherein the nucleic acid encodes a polypeptide having amylase activity;

96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, wherein the polypeptide has amylase activity.

Further preferred herein is an amylase comprising an amino acid sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO:54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID N0:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID N0 37.

Further preferred herein is a polypeptide having amylase activity (i.e., an amylase) comprising an amino acid sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54.

Further preferred herein is an amylase comprising an amino acid sequence that is at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,

SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

Further preferred herein is an amylase comprising an amino acid sequence that is at least 99% or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

Further preferred herein is an amylase comprising an amino acid sequence that is 99.5% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

Further preferred herein is an amylase comprising an amino acid sequence that is 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

Further preferred herein is an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

Further preferred herein is an amylase comprising an amino acid sequence that is at least

80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least

99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54.

Further preferred herein is an amylase comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54.

Further preferred herein is an amylase comprising one or more amino acid residue insertions, deletions, substitutions, or any combinations thereof to the amino acid sequence of: SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

Further preferred herein is an amylase comprising one or more amino acid residue substitution to the amino acid sequence of: SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, preferably SEQ ID NO: 54, preferably at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 positions, preferably, at 1-25 positions, more preferably, at 1-10 positions.

Further preferred herein is an amylase wherein the amino acid sequence is encoded by a polynucleotide having a nucleic acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full length polynucleotide sequence of SEQ ID NO: 55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38.

Further preferred herein is an amylase wherein the amino acid sequence is encoded by a polynucleotide having a nucleic acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full length polynucleotide sequence of SEQ ID NO:55.

Further preferred herein is an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO: 37, wherein the amylase has an increase in expression; activity; thermostability; stability; performance in laundry; and any combination thereof.

94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO: 37, wherein the amylase has an increase in thermostability. Further preferred herein is an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, wherein the amylase has an increase in thermostability.

94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO: 37, wherein the increase in thermostability is after a heat challenge at a temperature from 70 degrees C to 100 degrees C, preferably, wherein the increase in thermostability is after a heat challenge at a temperature from 70 degrees C to 90 degrees C, more preferably, between 75 degrees C to 85 degrees C, most preferably at 80 degrees C.

99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, wherein the increase in thermostability is after a heat challenge at a temperature from 70 degrees C to 100 degrees C, preferably, wherein the increase in thermostability is after a heat challenge at a temperature from 70 degrees C to 90 degrees C, more preferably, between 75 degrees C to 85 degrees C, most preferably at 80 degrees C.

Further preferred herein is an amylase comprising an amino acid sequence that is 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full- length amino acid sequence of: SEQ ID NO: 54, wherein amylase comprises an increase in thermostability after a heat challenge at 80 degrees C.

In another aspect the present invention refers to a composition comprising the polypeptide described herein. A composition may comprise combinations of the polypeptides with another enzyme. The combination of enzymes can be of the same class, for example a composition comprising a first amylase and a second amylase. Combinations of enzymes can be from a different class of enzymes, for example, a composition comprising a lipase and an amylase. Combinations of enzymes can be compositions comprising at least one amylase of the invention and one or more second enzymes. In one embodiment, the composition comprises one second enzyme, two second enzymes, three second enzymes, four second enzymes, or more than four second enzymes. In an embodiment, the second enzyme is selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a pectinase, and a nuclease, or any combination thereof.

A compositions comprising an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. In another embodiment, the composition further comprises a second enzyme selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a mannanase, a pectinase, xylanase, a nuclease, and any combination thereof. In preferred embodiment, the composition further comprises a second enzyme and the second enzyme is a different amylase. In another preferred embodiment, the composition further comprises a second enzyme and the second enzyme is a protease.

Preferably, a compositions comprising an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54. In another embodiment, the composition further comprises a second enzyme selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a mannanase, a pectinase, xylanase, a nuclease, and any combination thereof. In preferred embodiment, the composition further comprises a second enzyme and the second enzyme is a different amylase. In another preferred embodiment, the composition further comprises a second enzyme and the second enzyme is a protease.

Additional enzymes suitable for the hybrid or the composition of the present invention are further described below. In one embodiment, suitable enzymes include enzyme variants having enzymatic activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical when compared to the full length polypeptide sequence of the parent enzyme as disclosed below.

Amylase

Alpha-amylase (E.C. 3.2.1.1) enzymes may perform endohydrolysis of (l->4)-alpha-D- glucosidic linkages in polysaccharides containing three or more (l->4)-alpha-linked D-glucose units. Amylase enzymes act on starch, glycogen and related polysaccharides and oligosaccharides in a random manner; reducing groups are liberated in the alpha-configuration. Other examples of amylase enzymes include: Beta-amylase (E.C. 3.2.1.2), Glucan 1 ,4-alpha-maltotetraohydrolase (E.C. 3.2.1.60), Isoamylase (E.C. 3.2.1.68), Glucan 1 ,4-alpha-maltohexaosidase (E.C. 3.2.1.98), and Glucan 1 ,4-alpha-maltohydrolase (E.C. 3.2.1.133).

The amylases described below can be used as a parent amylase to create variant amylases by introducing one or more amino acid substitutions and / or deletions as described herein.

Many amylase enzymes have been described in patents and published patent applications including, but not limited to: WO 2002/068589, WO 2002/068597, WO 2003/083054, WO 2004/091544, and WO 2008/080093.

Amylases are known to be derived from Bacillus licheniformis having SEQ ID NO:2 as described in WO 95/10603. Suitable variants are those which are at least 90% identical to SEQ ID NO: 2 as described in WO 95/10603 and/or comprising one or more substitutions in the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444 which have amylolytic activity. Such variants are described in WO 94/02597, WO 94/018314, WO 97/043424 and SEQ ID NO:4 of WO 99/019467.

Amylases are known to be derived from B. stearothermophilus having SEQ ID NO: 6 as described in WO 02/10355 or an amylase which is at least 90% identical thereto having amylolytic activity with optionally having a C-terminal truncation over the wildtype sequence. Suitable variants of SEQ ID NO: 6 include those which is at least 90% identical thereto and/or further comprise a deletion in positions 181 and/or 182 and/or a substitution in position 193.

Amylases are known to be derived from Bacillus sp.707 having SEQ ID NO:6 as disclosed in WO 99/19467 or an amylase which is at least 90% identical thereto having amylolytic activity. Amylases are known from Bacillus halmapalus having SEQ ID NO:2 or SEQ ID NO:7 as described in WO 96/23872, also described as SP-722, or an amylase which is at least 90% identical to one of the sequences which has amylolytic activity.

Amylases are known to be derived from Bacillus sp. DSM 12649 having SEQ ID NO:4 as disclosed in WO 00/22103 or an amylase which is at least 90% identical thereto having amylolytic activity.

Amylases are known from Bacillus strain TS-23 having SEQ ID NO:2 as disclosed in WO 2009/061380 or an amylase which is at least 90 % identical thereto having amylolytic activity. Amylases are known from Cytophaga sp. having SEQ ID NO: 1 as disclosed in WO 2013/184577 or an amylase which is at least 90% identical thereto having amylolytic activity.

Amylases are known from Bacillus megaterium DSM 90 having SEQ ID NO: 1 as disclosed in WO 2010/104675 or an amylase which is at least 90% identical thereto having amylolytic activity.

Amylases are known having amino acids 1 to 485 of SEQ ID NO:2 as described in WO 00/60060 or amylases comprising an amino acid sequence which is at least 96% identical with amino acids 1 to 485 of SEQ ID NO:2 which have amylolytic activity.

Amylases are also known having SEQ ID NO: 12 as described in WO 2006/002643 or amylases having at least 80% identity thereto and have amylolytic activity. Suitable amylases include those having at least 80% identity compared to SEQ ID NO: 12 and/or comprising the substitutions at positions Y295F and M202LITV and have amylolytic activity.

Amylases are also known having SEQ ID NO:6 as described in WO 2011/098531 or amylases having at least 80% identity thereto having amylolytic activity. Suitable amylases include those having at least 80% identity compared to SEQ ID NO: 6 and/or comprising a substitution at one or more positions selected from the group consisting of 193 [G,A,S,T or M], 195 [F,W,Y,L,I or V], 197 [F,W,Y,L,I or V], 198 [Q or N], 200 [F,W,Y,L,I or V], 203 [F,W,Y,L,I or V], 206 [F,W,Y,N,L,I,V,H,Q,D or E], 210 [F,W,Y,L,I or V], 212 [F,W,Y,L,I or V], 213 [G,A,S,T or M] and 243 [F,W,Y,L,I or V] and have amylolytic activity.

Amylases are known having SEQ ID NO: 1 as described in WO 2013/001078 or amylases having at least 85% identity thereto having amylolytic activity. Suitable amylases include those having at least 85% identity compared to SEQ ID NO: 1 and/or comprising an alteration at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 and having amylolytic activity.

Amylases are known having SEQ ID NO:2 as described in WO 2013/001087 or amylases having at least 85% identity thereto and having amylolytic activity. Suitable amylases include those having at least 85% identity compared to SEQ ID NO:2 and/or comprising a deletion of positions 181+182, or 182+183, or 183+184 according to the numbering of SEQ ID NO: 2 as described in WO 2013/001087, which have amylolytic activity. Suitable amylases include those having at least 85% identity compared to SEQ ID NO:2 and/or comprising a deletion of positions 181+182, or 182+183, or 183+184 according to the numbering of SEQ ID NO: 2 as described in WO 2013/001087, which comprise one or two or more modifications in any of positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 and have amylolytic activity.

Amylases also include hybrid a-amylase from above mentioned amylases as for example as described m WO 2006/066594.

Commercially available amylase enzymes include: Duramyl™, Termamyl™, Termamyl SC™, Termamyl Ultra™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, Supramyl™ Amplify™, Amplify Prime™ and BAN™ (from Novozymes A/S), and Rapidase™, Purastar™, Purastar OxAm™ PoweraseTM, Effectenz™ (Ml 00 from DuPont), Preferenz™ (S 1000, SI 10 and FI 000; from DuPont), PrimaGreen™ (ALL; DuPont), Optisize™ (DuPont) and Kam™ (Kao) and Kemzyme™ (Biozym).

Lipase

“Lipases”, “lipolytic enzyme”, “lipid esterase”, all refer to an enzyme of EC class 3.1.1 (“carboxylic ester hydrolase”). Lipases (E.C. 3.1.1.3, Triacylglycerol lipase) may hydrolyze triglycerides to more hydrophilic mono- and diglycerides, free fatty acids, and glycerol. Lipase enzymes usually includes also enzymes which are active on substrates different from triglycerides or cleave specific fatty acids, such as Phospholipase A (E.C. 3.1.1.4), Galactolipase (E.C. 3.1.1.26), cutinase (EC 3.1.1.74), and enzymes having sterol esterase activity (EC 3.1.1.13) and/or wax-ester hydrolase activity (EC 3.1.1.50).

Many lipase enzymes have been described in patents and published patent applications including, but not limited to: W02000032758, W02003/089620, W02005/032496,

W02005/086900, W0200600976, W02006/031699, W02008/036863, WO2011/046812, and W02014059360.

Lipases are used in detergent and cleaning products to remove grease, fat, oil, and dairy stains. Commercially available lipases include but are not limited to: Lipolase™, Lipex™, Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (Gist-Brocades/ now DSM).

The methods for determining lipolytic activity are well-known in the literature (see e.g. Gupta et al. (2003), Biotechnol. Appl. Biochem. 37, p. 63-71). E.g. the lipase activity may be measured by ester bond hydrolysis in the substrate para-nitrophenyl palmitate (pNP-Palmitate, C: 16) and releases pNP which is yellow and can be detected at 405 nm.

Protease

Enzymes having proteolytic activity are called “proteases” or “peptidases”. Proteases are active proteins exerting “protease activity” or “proteolytic activity”.

Proteases are members of class EC 3.4. Proteases include aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl-peptidases and tripeptidyl-peptidases (EC 3.4.14), peptidyl- dipeptidases (EC 3.4.15), serine-type carboxypeptidases (EC 3.4.16), metallocarboxypeptidases (EC 3.4.17), cysteine-type carboxypeptidases (EC 3.4.18), omega peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metallo-endopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25), endopeptidases of unknown catalytic mechanism (EC 3.4.99).

Commercially available protease enzymes include but are not limited to Lavergy™ Pro (BASF); Alcalase®, Blaze®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect® Prime, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Ultimase®, Opticlean®, Effectenz®, Preferenz® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N. V.), Bacillus lentus Alkaline Protease, and KAP ( Bacillus alkalophilus subtilisin) from Kao.

At least one protease may be selected from serine proteases (EC 3.4.21). Serine proteases or serine peptidases (EC 3.4.21) are characterized by having a serine in the catalytically active site, which forms a covalent adduct with the substrate during the catalytic reaction. A serine protease may be selected from the group consisting of chymotrypsin (e.g., EC 3.4.21.1), elastase (e.g., EC 3.4.21.36), elastase (e.g., EC 3.4.21.37 or EC 3.4.21.71), granzyme (e.g., EC 3.4.21.78 or EC 3.4.21.79), kallikrein (e.g., EC 3.4.21.34, EC 3.4.21.35, EC 3.4.21.118, or EC 3.4.21.119,) plasmin (e.g., EC 3.4.21.7), trypsin (e.g., EC 3.4.21.4), thrombin (e.g., EC 3.4.21.5,) and subtilisin (also known as subtilopeptidase, e.g., EC 3.4.21.62), the latter hereinafter also being referred to as “subtilisin”.

Cellulase

"Cellulases", “cellulase enzymes” or “cellulolytic enzymes” are enzymes involved in hydrolysis of cellulose. Three major types of cellulases are known, namely endo-ss-l,4-glucanase (endo-l,4-P-D-glucan 4-glucanohydrolase, E.C. 3.2.1.4; hydrolyzing b- 1 ,4-glucosidic bonds in cellulose), cellobiohydrolase (1,4-P-D-glucan cellobiohydrolase, EC 3.2.1.91), and ss-glucosidase (EC 3.2.1.21).

Cellulase enzymes have been described in patents and published patent applications including, but not limited to: WO1997/025417, WO 1998/024799, W02003/068910, W02005/003319, and W02009020459.

Commercially available cellulase enzymes include are Celluzyme™, Endolase™, Carezyme™, Cellusoft™, Renozyme™, Celluclean™ (from Novozymes A/S), Ecostone™, Biotouch™, Econase™, Ecopulp™ (from AB Enzymes Finland), Clazinase™, and Puradax HA™, Genencor detergent cellulase L, IndiAge™ Neutra (from Genencor International Inc./DuPont), Revitalenz™ (2000 from DuPont), Primafast™ (DuPont) and KAC-500™ (from Kao Corporation).

Cellulases according to the invention have “cellulolytic activity” or “cellulase activity”. Assays for measurement of cellulolytic activity are known to those skilled in the art. For example, cellulolytic activity may be determined by cellulase hydrolyses carboxymethyl cellulose to reducing carbohydrates, the reducing ability of which is determined colorimetrically by means of the ferricyanide reaction, according to Hoffman, W. S., J. Biol. Chem. 120, 51 (1937).

Mannanase Mannanase (E.C. 3.2.1.78) enzymes hydrolyze internal b-1,4 beta-D-mannosidic linkages in mannans, galactomannans and glucomannans. “Mannanase” may be an alkaline mannanase of Family 5 or 26. Mannanase are useful components of washing and/or cleaning formulations since mannanase remove part of hemicellulose containing stains. Insufficient removal of these types of stains may e.g. result in fabric graying. The major constituents of hemicellulose are hetero-l,4-D- xylans and herto-1 ,4-beta-mannans. Mannans are polysaccharides with a backbone of b-l ,4-linked D-mannopyranosyl residues, which can contain galactose or acetyl substitutions and may have glucose residues in the backbone. Mannanase enzymes are known to be derived from wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 99/064619. Commercially available mannanase enzymes include: Mannaway® (Novozymes AIS).

Pectate lyase

Pectate lyase (E.C. 4.2.2.2) enzymes eliminative cleavage of (l->4)-alpha-D-galacturonan to give oligosaccharides with 4-deoxy-alpha-D-galact-4-enuronosyl groups at their non-reducing ends. Pectate lyase enzymes have been described in patents and published patent applications including, but not limited to: W02004/090099. Pectate lyase are known to be derived from Bacillus, particularly B. licheniformis or B. agaradhaerens, or a variant derived of any of these, e.g. as described in US 6,124,127, WO 99/027083, WO 99/027084, WO 2002/006442, WO 2002/092741, WO 2003/095638. Commercially available pectate lyase enzymes include: Xpect™, Pectawash™ and

Pectaway™ (Novozymes A/S); PrimaGreen™, EcoScour (DuPont).

Nuclease

Nuclease (EC 3.1.21.1) also known as Deoxyribonuclease I, or DNase preforms endonucleolytic cleavage to 5'-phosphodinucleotide and 5'-phosphooligonucleotide end-products. Nuclease enzymes have been described in patents and published patent applications including, but not limited to: US3451935, GB1300596, DE10304331, WO2015155350, WO20 15155351, WO2015166075, WO2015181287, and WO2015181286.

In one aspect of the invention, at least one amylase variant of the invention is provided in combination with at least one protease. In one embodiment, an amylase variant of the invention is stable in the presence of at least one protease. In one embodiment, an amylase variant of the invention has increased protease stability when compared to the respective amylase parent. In one embodiment, at least one protease is selected from subtilisin 309 as disclosed as sequence a) in Table I of WO 89/06279 or a variant thereof which is at least 80% identical thereto and has proteolytic activity. In one embodiment, an amylase variant of the invention has increased protease stability in the presence of said subtilisin 309 or a variant thereof which is at least 80% identical thereto when compared to the amylase according to SEQ ID NO: 1.

The protease may itself be stabilized by a protease stabilizer or the protease may be non- stabilized. In one embodiment, an amylase variant of the invention has increased protease stability in the presence of a non-stabilized subtilisin 309 or a non-stabilized variant thereof which is at least 80% identical thereto, when compared to the amylase according to SEQ ID NO: 1.

Methods of making:

In another embodiment, the present invention refers to a method of making the variant polypeptide as described herein, comprising: providing a nucleic acid sequence encoding the polypeptide described herein, transforming the nucleic acid sequence into a host cell, cultivating the host cell to produce the variant polypeptide, and optionally purifying the variant polypeptide from the host cell.

A polynucleotide encoding a polypeptide may be “expressed”. The term “expression” or “gene expression” means the transcription of a specific gene or specific genes or specific nucleic acid construct. The term “expression” or “gene expression” means the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.

Industrial production of enzymes usually is done by using expression systems. “Expression system” may mean a host microorganism, expression hosts, host cell, production organism, or production strain and each of these terms can be used interchangeably. In one embodiment, the expression host is selected from the group consisting of: a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system. The expression host may be a wildtype cell or a recombinant cell. “Wild-type cells” herein means cells prior to a certain modification. The term “recombinant cell” (also called “genetically modified cell” herein) refers to a cell which has been genetically altered, modified or engineered such it that exhibits an altered, modified or different genotype as compared to the wild-type cell which it was derived from. The “recombinant cell” may comprise an exogenous polynucleotide encoding a certain protein or enzyme and therefore may express said protein or enzyme.

Thus, in one embodiment, the invention is directed to a genetic construct comprising a polynucleotide encoding the amylase as described herein.

In one embodiment, the invention is directed to an expression vector comprising a polynucleotide encoding the amylase as described herein.

In one embodiment, the invention is directed to a host cell comprising a polynucleotide encoding the amylase as described herein.

In yet another embodiment, the present invention is directed to a method of expressing a polynucleotide, comprising the steps of

(a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding the amylase described herein by introducing the nucleic acid construct comprising the polynucleotide encoding the amylase as described herein into the host cell;

(b) cultivating the recombinant host cell of step (a) under conditions conductive for the expression of the polynucleotide; and

(c) optionally, recovering a protein of interest encoded by the polynucleotide.

Examples of expression systems include but are not limited to: Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, fusarium oxysporum, Fusarium heterosporum, Escherichia coli, Bacillus, preferably Bacillus pumilus, Bacillus subtilis, or Bacillus licheniformis, Pseudomonas, preferably Pseudomonas fluorescens, Pichia pastoris (also known as Komagataella phaffii), Myceliopthora thermophile (Cl), Themothelomyces thermophila, Schizosaccharomyces pombe, Trichoderma, preferably Trichoderma reesei and Saccharomyces, preferably Saccharomyces cerevisiae. The variant polypeptides may be produced using the expression system listed above. In one embodiment, the bacterial expression system is selected from an E. coli, a Bacillus, a Pseudomonas, and a Streptomyces. In one embodiment, the yeast expression system is selected from a Candida, aPichia, a Saccharomyces, and/or a Schizosaccharomyces. In one embodiment, the fungal expression system is selected from a Penicillium, an Aspergillus, a Fusarium, a Myceliopthora, a Rhizomucor, a Rhizopus, a Thermomyces, and a Trichoderma.

The term "heterologous” (or exogenous or foreign or recombinant) in the context of polynucleotides and polypeptides is defined herein as:

(a) not native to the host cell;

(b) native to the host cell but structural modifications, e.g., deletions, substitutions, and/or insertions, are included as a result of manipulation of the DNA of the host cell by recombinant DNA techniques to alter the native sequence; or

(c) native to the host cell but expression is quantitatively altered, or expression is directed from a genomic location different from the native host cell as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, e.g., a stronger promoter.

With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous” is used to characterize that the two or more polynucleotide sequences or two or more amino acid sequences do not occur naturally in the specific combination with each other.

“Genetic construct” or “expression cassette” as used herein, is a DNA molecule composed of at least one sequence of interest to be expressed, operably linked to one or more control sequences (at least to a promoter) as described herein. Typically, the expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. Additional regulatory elements may include transcriptional as well as translational enhancers. An intron sequence may also be added to the 5' untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol. The expression cassette may be part of a vector or may be integrated into the genome of a host cell and replicated together with the genome of its host cell. The expression cassette usually is capable of increasing or decreasing expression.

The term “vector” as used herein comprises any kind of construct suitable to carry foreign polynucleotide sequences for transfer to another cell, or for stable or transient expression within a given cell. The term “vector” as used herein encompasses any kind of cloning vehicles, such as but not limited to plasmids, phagemids, viral vectors (e.g., phages), bacteriophage, baculoviruses, cosmids, fosmids, artificial chromosomes, or and any other vectors specific for specific hosts of interest. Low copy number or high copy number vectors are also included. Foreign polynucleotide sequences usually comprise a coding sequence which may be referred to herein as “gene of interest”. The gene of interest may comprise introns and exons, depending on the kind of origin or destination of host cell.

A vector as used herein may provide segments for transcription and translation of a foreign polynucleotide upon transformation into a host cell or host cell organelles. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication that is required for its maintenance and/or replication in a specific cell type, one or more selectable markers, a polyadenylation signal, a suitable site for the insertion of foreign coding sequences such as a multiple cloning site etc. One example is when a vector is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Non-limiting examples of suitable origins of replication include the fl-ori and colEl.

A vector may replicate without integrating into the genome of a host cell, e.g. as a plasmid in a bacterial host cell, or it may integrate part or all of its DNA into the genome of the host cell and thus lead to replication and expression of its DNA.

Foreign nucleic acid may be introduced into a vector by means of cloning. Cloning may mean that by cleavage of the vector (e.g. within the multiple cloning site) and the foreign polynucleotide by suitable means and methods (e.g., restriction enzymes), fitting structures within the individual nucleic acids may be created that enable the controlled fusion of said foreign nucleic acid and the vector.

Once introduced into the vector, the foreign nucleic acid comprising a coding sequence may be suitable to be introduced (transformed, transduced, transfected, etc.) into a host cell or host cell organelles. A cloning vector may be chosen suitable for expression of the foreign polynucleotide sequence in the host cell or host cell organelles.

The term “introduction” or “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. That is, the term “transformation” as used herein is independent from vector, shuttle system, or host cell, and it not only relates to the polynucleotide transfer method of transformation as known in the art (cf , for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but it encompasses any further kind polynucleotide transfer methods such as, but not limited to, transduction or transfection. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.

In one embodiment of the invention, a vector is used for transformation of a host cell.

The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. “Stable transformation” may mean that the transformed cell or cell organelle passes the nucleic acid comprising the foreign coding sequence on to the next generations of the cell or cell organelles. Usually stable transformation is due to integration of nucleic acid comprising a foreign coding sequence into the chromosomes or as an episome (separate piece of nuclear DNA).

“Transient transformation” may mean that the cell or cell organelle once transformed expresses the foreign nucleic acid sequence for a certain time - mostly within one generation. Usually transient transformation is due to nucleic acid comprising a foreign nucleic acid sequence is not integrated into the chromosomes or as an episome.

Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

Recombinant cells may exhibit “increased” or “decreased” expression when compared to the respective wild-type cell.

The term “increased expression”, “enhanced expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level (which can be absence of expression or immeasurable expression as well). Reference herein to “increased expression”, “enhanced expression” or “overexpression” is taken to mean an increase in gene expression and/or, as far as referring to polypeptides, increased polypeptide levels and/or increased polypeptide activity, relative to control organisms. The increase in expression may be in increasing order of preference at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or 100% or even more compared to that of control organisms. Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non- heterologous form of a polynucleotide so as to increase expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see Kmiec et al., US 5,565,350; Zarling et al., WO 93/22443), or isolated promoters may be introduced into an organism in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.

An intron sequence may also be added to the 5' untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis etal. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit.

To obtain increased expression or overexpression of a polypeptide most commonly the nucleic acid encoding this polypeptide is overexpressed in sense orientation with a polyadenylation signal. Introns or other enhancing elements may be used in addition to a promoter suitable for driving expression with the intended expression pattern.

Enzymes are generally produced commercially by using recombinant cells which express the desired enzyme by cultivation of the same under conditions suitable for expression of the desired enzyme.

Cultivation normally takes place in a suitable nutrient medium allowing the recombinant cells to grow (this process may be called fermentation) and express the desired protein. At the end of fermentation, fermentation broth is collected and may be further processed, wherein the fermentation broth comprises a liquid fraction and a solid fraction.

The enzyme of interest may be further purified from the fermentation broth. The term “purification” or “purifying” refers to a process in which at least one component, e.g., a protein of interest, is separated from at least another component, e.g., a particulate matter of a fermentation broth, and transferred into a different compartment or phase, wherein the different compartments or phases do not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filtration membrane or cloth, i.e., filtrate and retentate; examples of such different phases are pellet and supernatant or cake and filtrate, respectively. The resulting solution after purifying the enzyme of interest from the fermentation broth is called herein “purified enzyme solution”.

The desired enzyme may be secreted (into the liquid fraction of the fermentation broth) or may not be secreted from the host cells (and therefore is comprised in the cells of the fermentation broth). Depending on this, the desired enzyme may be recovered from the liquid fraction of the fermentation broth or from cell lysates. Recovery of the desired enzyme uses methods known to those skilled in the art. Suitable methods for recovery of proteins or enzymes from fermentation broth include but are not limited to collection, centrifugation, filtration, extraction, and precipitation. If the enzyme of interest precipitates or crystallizes in the fermentation broth or binds at least in part to the particulate matter of the fermentation broth additional treatment steps might be needed to release the enzyme from the biomass or solubilize enzyme crystals and precipitates. US6316240B1 describes a method for recovering an enzyme, which precipitates and / or crystallizes during fermentation, from the fermentation broth. In case the desired enzyme is comprised in the cells of the fermentation broth release of the enzyme from the cells might be needed. Release from the cells can be achieved for instance, but not being limited thereto, by cell lysis with techniques well known to the skilled person.

The purified enzyme solution may be further processed to form an “enzyme formulation”. “Enzyme formulation” means any non-complex formulation comprising a small number of ingredients, wherein the ingredients serve the purpose of stabilizing the enzymes comprised in the enzyme formulation and/or the stabilization of the enzyme formulation itself. The term “enzyme stability” relates to the retention of enzymatic activity as a function of time during storage or operation. The term “enzyme formulation stability” relates to the maintenance of physical appearance of the enzyme formulation during storage or operation as well as the avoidance of microbial contamination during storage or operation.

An “enzyme formulation” is a composition which is meant to be formulated into a complex formulation which itself may be determined for final use. An “enzyme formulation” according to the invention is not a complex formulation comprising several components, wherein the components are formulated into the complex formulation to exert each individually a specific action in a final application. A complex formulation may be without being limited thereto a detergent formulation, wherein individual detergent components are formulated in amounts effective in the washing performance of the detergent formulation.

In one aspect of the invention, at least one amylase variant of the invention is comprised in an enzyme formulation.

The enzyme formulation can be either solid or liquid. Enzyme formulations can be obtained by using techniques known in the art. For instance, without being limited thereto, solid enzyme formulations can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described for instance in W09419444A1 and W09743482A1.

“Liquid” in the context of enzyme formulation is related to the physical appearance at 20°C and 101.3 kPa.

Liquid enzyme formulations may comprise amounts of enzyme in the range of 0.1% to 40% by weight, or 0.5% to 30% by weight, or 1% to 25% by weight, or 3% to 10%, all relative to the total weight of the enzyme formulation.

The liquid enzyme formulation may comprise more than one type of enzyme. In one embodiment, the enzyme formulation comprises one or more amylases according to the present invention. In one embodiment, the enzyme formulation comprises one or more amylases according to the present invention and at least one additional enzyme selected from the group consisting of selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a pectinase, a nuclease, and any combination thereof.

Aqueous enzyme formulations of the invention may comprise water in amounts of more than about 50% by weight, more than about 60% by weight, more than about 70% by weight, or more than about 80% by weight, all relative to the total weight of the enzyme formulation.

Liquid enzyme formulations of the invention may comprise residual components such as salts originating from the fermentation medium, cell debris originating from the production host cells, metabolites produced by the production host cells during fermentation.

In one embodiment, residual components may be comprised in liquid enzyme formulations in amounts less than 30% by weight, less than 20% by weight less, than 10% by weight, or less than 5% by weight, all relative to the total weight of the aqueous enzyme formulation. In one embodiment, the enzyme formulation, in particular the liquid enzyme formulation, comprises in addition to the one or more enzymes one or more additional compounds selected from the group consisting of solvent, salt, pH regulator, preservative, stabilizer, chelators, and thickening agent. The preservative in a liquid enzyme formulation maybe a sorbitol, a benzoate, a proxel, or any combination therefore. The stabilizers in a liquid enzyme formulation maybe an MPG, a glycerol, an acetate, or any combination thereof. The chelators in a liquid enzyme formulation maybe a citrate.

In one embodiment, an enzyme formulation comprises at least one polypeptide variant of the invention and at least one preservative. Non-limiting examples of suitable preservatives include (quaternary) ammonium compounds, isothiazolinones, organic acids, and formaldehyde releasing agents. Non-limiting examples of suitable (quaternary) ammonium compounds include benzalkonium chlorides, polyhexamethylene biguanide (PHMB), Didecyldimethylammonium chloride (DDAC), and N-(3-aminopropyl)-N-dodecylpropane-l, 3-diamine (Diamine). Non limiting examples of suitable isothiazolinones include l,2-benzisothiazolin-3-one (BIT), 2- methyl-2H-isothiazol-3 -one (MIT), 5-chloro-2-methyl-2H-isothiazol-3-one (CIT), 2-octyl-2H- isothiazol-3-one (OIT), and 2-butyl-benzo[d]isothiazol-3-one (BBIT). Non-limiting examples of suitable organic acids include benzoic acid, sorbic acid, L-(+)-lactic acid, formic acid, and salicylic acid. Non-limiting examples of suitable formaldehyde releasing agent include N,N'- methylenebismorpholine (MBM), 2,2',2"-(hexahydro-l,3,5-triazine-l,3,5- triyl)triethanol (HHT), (ethylenedioxy)dimethanol, .alpha., .alpha. ',. alpha "-trimethyl-l, 3, 5-triazine-l, 3, 5(2H,4H,6H)- triethanol (HPT), 3,3'-methylenebis[5-methyloxazolidine] (MBO), and cis-l-(3-chloroallyl)- 3,5,7-triaza-l- azoniaadamantane chloride (CTAC).

Further useful preservatives include iodopropynyl butylcarbamate (IPBC), halogen releasing compounds such as dichloro-dimethyl-hydantoine (DCDMH), bromo-chloro-dimethyl- hydantoine (BCDMH), and dibromo-dimethyl-hydantoine (DBDMH); bromo-nitro compounds such as Bronopol (2-bromo-2-nitropropane-l,3-diol), 2,2-dibromo-2-cyanoacetamide (DBNPA); aldehydes such as glutaraldehyde; phenoxy ethanol; Biphenyl-2-ol; and zinc or sodium pyrithione.

In one embodiment, an enzyme formulation comprises at least one polypeptide variant of the invention and at least one enzyme stabilizer. An enzyme stabilizer is selected from substances which are capable of reducing loss of enzymatic activity during storage of at least one enzyme comprised in a liquid enzyme formulation. Reduced loss of enzymatic activity within this invention may mean that the loss of enzymatic activity is reduced by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% when compared to the initial enzymatic activity before storage. Preferred stabilizers are selected from the group consisting of salt (e.g., CaC12), propanediol, polyethylene glycol, an MPG, a glycerol, an acetate, or any combination thereof.

Enzyme applications

In another embodiment, the polypeptide variant as described herein may be used in foods, for example the enzyme can be an additive for baking. The enzymes can be used in feed, for example the enzyme is an animal feed additive. The enzyme can be used in the starch processing industry, for example the amylases are used in the conversion of starch to ethanol or sugars (high fructose corn syrup) and other byproducts such as oil, dry distiller’s grains, etc. The polypeptide variants are used in in pulp and paper processing, for example, the enzymes can be used for improving paper strength. The enzymes can be used for mining and oil well services, for example cellulases can be used for breaking guar during oil well fracturing. In one embodiment, the polypeptide variant as described herein are used in detergent formulations or cleaning formulations.

In one embodiment, the present invention refers to a method of preparing a dough or a baked product prepared from the dough, the method comprising adding one of the variant polypeptides having amylase activity as described herein to the dough and baking it. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for processing starch. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for cleaning or washing textiles, hard surfaces, or dishes. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for making ethanol. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for processing pulp or paper. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for feeding an animal.

In one embodiment, the amylases of the present invention are used in detergent formulations or cleaning formulations. “Detergent formulation” or “cleaning formulation” means compositions designated for cleaning soiled material. Cleaning includes laundering and hard surface cleaning. Soiled material according to the invention includes textiles and/or hard surfaces.

The term “laundering” relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a detergent composition of the present invention. The laundering process may be carried out by using technical devices such as a household or an industrial washing machine. Alternatively, the laundering process may be done by hand.

The term “textile” means any textile material including yarns (thread made of natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, as well as fabrics (a textile made by weaving, knitting or felting fibers) made of these materials such as garments (any article of clothing made of textile), cloths and other articles.

The term “fibers” includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are of plant (such as flax, jute and cotton) or animal origin, comprising proteins like collagen, keratin and fibroin (e.g. silk, sheep wool, angora, mohair, cashmere). Examples for fibers of synthetic origin are polyurethane fibers such as Spandex® or Lycra®, polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. Fibers may be single fibers or parts of textiles such as knitwear, woven, or nonwovens.

The term “hard surface cleaning” is defined herein as cleaning of hard surfaces wherein hard surfaces may include any hard surfaces in the household, such as floors, furnishing, walls, sanitary ceramics, glass, metallic surfaces including cutlery or dishes.

The term “dish wash” refers to all forms of washing dishes, e.g. by hand or automatic dish wash. Dish washing includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics such as melamine, metals, china, glass and acrylics.

The detergent formulation of the invention comprises one or more detergent component(s). The component(s) chosen depend(s) on the desired cleaning application and/or physical form of a detergent composition.

The term “detergent component” is defined herein to mean any types of ingredient, which is suitable for detergent compositions, such as surfactants, building agents, polymers, bleaching systems. Any component(s) known in the art acknowledging their known characteristics are suitable detergent component(s) according to the invention. Detergent components in one embodiment means components which provide washing or cleaning performance, or which effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desiccants) when present in effective amounts.

Usually, a detergent composition is a complex formulation of more than two detergent components.

Detergent components may have more than one function in the final application of a detergent formulation, therefore any detergent component mentioned in the context of a specific function herein, may also have another function in the final application of a detergent formulation. The function of a specific detergent component in the final application of a detergent formulation usually depends on its amount within the detergent formulation, i.e. the effective amount of a detergent component.

The term “effective amount” includes amounts of certain components to provide effective stain removal and effective cleaning conditions (e.g. pH, quantity of foaming), amounts of certain components to effectively provide optical benefits (e.g. optical brightening, dye transfer inhibition), and amounts of certain components to effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desiccants).

In one embodiment, a detergent formulation is a formulation of more than two detergent components, wherein at least one component is effective in stain-removal, at least one component is effective in providing the optimal cleaning conditions, and at least one component is effective in maintaining the physical characteristics of the detergent.

Cleaning performance is evaluated under relevant cleaning conditions. The term "relevant cleaning conditions" herein refers to the conditions, particularly cleaning temperature, time, cleaning mechanics, suds concentration, type of detergent and water hardness, actually used in laundry machines, automatic dish washers or in manual cleaning processes.

Individual detergent components and usage in detergent compositions are known to those skilled in the art. Suitable detergent components comprise inter alia surfactants, builders, polymers, alkaline, bleaching systems, fluorescent whitening agents, suds suppressors and stabilizers, hydrotropes, and corrosion inhibitors. Further examples are described e.g. in “complete Technology Book on Detergents with Formulations (Detergent Cake, Dishwashing Detergents, Liquid & Paste Detergents, Enzyme Detergents, Cleaning Powder & Spray Dried Washing Powder)”, Engineers India Research Institute (EIRI), 6^th edition (2015). Another reference book for those skilled in the art may be “Detergent Formulations Encyclopedia”, Solverchem Publications, 2016.

Detergent components vary in type and/or amount in a detergent formulation depending on the desired application such as laundering white textiles, colored textiles, and wool. The component(s) chosen further depend(s) on physical form of a detergent formulation (liquid, solid, gel, provided in pouches or as a tablet, etc.). The component(s) chosen e.g. for laundering formulations further depend on regional conventions which themselves are related to aspects like washing temperatures used, mechanics of laundry machine (vertical vs. horizontal axis machines), water consumption per wash cycle etc. and geographical characteristics like average hardness of water.

For example: A low detergent concentration system includes laundering formulations where less than about 800 ppm of detergent components are present in the wash water. A medium detergent concentration includes laundering formulations where between about 800 ppm and about 2,000 ppm of detergent components are present in the wash water. A high detergent concentration includes laundering formulations where more than about 2,000 ppm of detergent components are present in the wash water.

The numeric ranges recited for the individual detergent components provide amounts comprised in detergent compositions. Such ranges have to be understood to be inclusive of the numbers defining the range and include each integer within the defined range.

If not described otherwise, “% by weight” or “% w/w” is meant to be related to total detergent composition. In this case “% by weight” or “% w/w” is calculated as follows: concentration of a substance as the weight of that substance divided by the total weight of the composition, multiplied by 100.

Detergent formulations of the invention may comprise one or more surfactant(s). "Surfactant" (synonymously used herein with “surface active agent”) means an organic chemical that, when added to a liquid, changes the properties of that liquid at an interface. According to its ionic charge, a surfactant is called non-ionic, anionic, cationic, or amphoteric. Non-limiting examples of surfactants are disclosed McCutcheon's 2016 Detergents and Emulsifiers, and McCutcheon's 2016 Functional Materials, both North American and International Edition, MC Publishing Co, 2016 edition. Further useful examples are disclosed in earlier editions of the same publications which are known to those skilled in the art.

Non-ionic surfactant means a surfactant that contains neither positively nor negatively charged (i.e. ionic) functional groups. In contrast to anionic and cationic surfactants, non-ionic surfactants do not ionize in solution.

Preferred methods and uses:

Preferred herein is a method of making the amylase comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44; comprising: providing a nucleic acid sequence encoding said amylase; transforming the nucleic acid sequence into an expression host, cultivating the expression host to produce the amylase, and optionally purifying the amylase.

Preferred herein is a method of making the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; comprising: providing a nucleic acid sequence comprising: SEQ ID NO: 55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; transforming the nucleic acid sequence into an expression host, cultivating the expression host to produce the amylase, and optionally purifying the amylase.

Preferred herein is a method of making the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54; comprising: providing a nucleic acid sequence comprising: SEQ ID NO:55; transforming the nucleic acid sequence into an expression host, cultivating the expression host to produce the amylase, and optionally purifying the amylase.

The method of making the amylase wherein the expression host is selected from the group consisting of: a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system.

The method of making the amylase wherein the bacterial expression system is selected from an E. coli, a Bacillus, a Pseudomonas, and a Streptomyces, wherein the yeast expression system is selected from a Candida, a Pichia, a Saccharomyces, a Schizosaccharomyces or, wherein the fungal expression system is selected from a Penicillium, an Aspergillus, a Fusarium, a Myceliopthora, a Themothelomyces, a Rhizomucor, a Rhizopus, a Thermomyces, and a Trichoderma, preferably Bacillus.

Preferred herein is a method of making the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; wherein the expression host is a Bacillus host cell, preferably selected from Bacillus pumilus, Bacillus subtilis, and Bacillus licheniformis, most preferably, Bacillus licheniformis.

Preferred herein is a method of making the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, wherein the expression host is a Bacillus host cell, preferably selected from Bacillus pumilus, Bacillus subtilis, and Bacillus licheniformis, most preferably, Bacillus licheniformis.

Preferred herein is a method of use of a C domain of a first amylase said C domain having an amino acid sequence which has at least 75% identity to the amino acid sequence of SEQ ID NO: 44 for improving one or more properties selected from the group consisting of stability, pH profile, expression, activity, thermostability, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, performance in laundry, processing starch, cleaning textiles, cleaning hard surfaces, cleaning dishes, making ethanol, processing pulp or paper, and feeding an animal of a second alpha amylase having an A and B domain with at least 75 % identity to the amino acid sequence of SEQ ID NO: 42 said use comprising replacing the C domain of the second alpha-amylase with the C domain of the first alpha-amylase.

Preferred herein is a method of preparing a dough or a baked product prepared from the dough, the method comprising adding the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l l, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, to the dough and baking it.

Preferred herein is a method of preparing a dough or a baked product prepared from the dough, the method comprising adding the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54 to the dough and baking it.

Preferred herein is a method of use of the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, for processing starch, for cleaning or washing textiles, hard surfaces, or dishes, for making ethanol, for processing pulp or paper, or for feeding an animal, preferably, for cleaning or washing textiles, hard surfaces, or dishes.

Preferred herein is a method of use of the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54 for processing starch, for cleaning or washing textiles, hard surfaces, or dishes, for making ethanol, for processing pulp or paper, or for feeding an animal.

Preferred herein is a method of use of the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO: 37, for cleaning or washing textiles, hard surfaces, or dishes.

Preferred herein is a method of use of the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54 for cleaning or washing textiles, hard surfaces, or dishes. Preferred herein is a method of use of the amylase comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO:54 for cleaning or washing textiles, hard surfaces, or dishes.

Preferred herein is a method of use of the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO: 37, for cleaning or washing textiles.

Preferred herein is a method of use of the amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of: SEQ ID NO: 54 for cleaning or washing textiles.

Preferred herein is a method of use of the amylase comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54 for cleaning or washing textiles.

Examples

Example 1 Generation of amylase expression constructs

The genes coding for the amino acid sequences were codon optimized to match the native codon abundance of Bacillus subtilis, and physically synthesized by an external, commercial DNA provider. Upon receipt, these genes were cloned by restriction-ligation based standard protocols into a gram-positive expression vector featuring regions coding for a promoter, a secretion signal peptide, and a ribosome binding site that are known to provide expression in the Bacillaceae family of organisms (e.g. the promoter driving expression of the B. subtilis amyE gene, the signal peptide of B. subtilis YdjM enzyme, and a consensus Shine-Dalgarno sequence). The vector furthermore contained an antibiotic based selection marker and an origin of replication.

Post reaction, the plasmid assembly mixtures were transformed according to the method of Spizizen (Anagnostopoulos,C. and Spizizen, J. (1961). J. Bacteriol. 81, 741-746.) into a B. subtilis PY79 KO- 7S (Bacillus Genetic Stock Center, BGSCID:1S145; Zeigler D.R) derivative named Bs#056 with an integrated DNA-methyltransferase gene as described for B. subtilis Bs#053 in W02019016051. Successful transformations were selected by plating on LB agar plates supplemented with 20 pg/ml kanamycin sulfate and incubating overnight at 37 degrees C. After the overnight selection, individual colonies were obtained which were then grown in a rich medium (e.g. LB broth) with 20 pg/ml kanamycin sulfate overnight at 37 degrees with shaking at 250 rpm. The following morning, cells were pelleted by centrifugation, and plasmid DNA was isolated by alkaline lysis method with a Macherey-Nagel NucleoSpin kit. The isolated DNA could then be transformed into electrocompetent Bacillus licheniformis cells.

Preparation of electrocompetent B. licheniformis cells and transformation of DNA was performed as essentially described by Brigidi et al (Brigidi,P., Mateuzzi,D. (1991). Biotechnol. Techniques 5, 5) with the following modification: Upon transformation of DNA, cells are recovered in 1ml LBSPG buffer and incubated for 60min at 37°C (Vehmaanpera J., 1989, LEMS Microbio. Lett., 61: 165-170) following plating on selective LB-agar plates (20 pg/ml kanamycin sulfate) and incubating overnight at 37 degrees C.

Example 2: Amylase expression and protein quantification

Single colonies of the expression strains were picked into 60 pL of rich medium (e.g. LB broth) supplemented with 20 pg/mL kanamycin sulfate in a 96- well plate (GE Life Sciences part 28403943). The cultures were grown at 37 degrees C for 16 hours, then 15 pL of culture was used to inoculate 600 pL of defined glucose-mineral media and 20 pg/mL kanamycin sulfate in a 96 well plate. The cultures were grown at 37 degrees C for 48 hours, after which the supernatant was harvested by pelleting the cells with centrifugation and removing the residual culture liquid. The expression levels (concentration in mg/mL) of variant polypeptide hybrids having alpha-amylase activity were identified by LabChip (LC) analysis and corresponding expression plasmids were sequenced for verification. Example 3 : Red starch and residual activity assays

Quantitation of starch hydrolysis for the variant polypeptides containing alpha-amylase activity was measured using the Red Starch method as described by Megazyme, “Assay of Alpha- Amylase using Red-Starch” with the following modifications. 10 pL of 1.33% red starch prepared in 50 mM HEPES, pH 8.0 buffer was reacted with 10 pL enzyme diluted in 50 mM HEPES, pH 8.0 buffer at 25°C. The reaction was terminated after 10 minutes by the addition of 50pL of 200 proof ethanol. After vigorous mixing, the reaction was equilibrated for 10 minutes at room temperature followed by centrifugation at 1,200 x g for 10 minutes. 40 pL of the reaction was transferred and the solution absorption was read at 510 nm in a BioTek plate reader. Residual activity was calculated by comparing the activity of each enzyme as measured using the red starch assay before and after a heat challenge. A heat challenge was conducted on enzyme diluted in 50 mM HEPES, pH 8.0 buffer by first heating the sample to 80 degrees C for 15 minutes, chilling at 4 degrees C for 10 minutes, then holding at 24 degrees C for 5 minutes before testing using the red starch assay at 25 degrees C.

Each enzyme/temperature pair was done in technical duplicates (25°C done in triplicate or n=5), with two independent experiments run on different days. For residual activity plots, the reference for "100% activity" was the highest activity measurement across all temperatures. The result for the 80 °C heat challenge is shown in Figure 1. It is clearly visible that replacing the C domain of SEQ ID NO:41 with the C domain of SEQ ID NO:40 resulting in an amylase having SEQ ID NO: 54 (with additional mutations at the positions 430 and 454) greatly increases the thermostability. Using closely related C domains (like closely related to the C domain of SEQ ID NO: 40) gives rise to hybrids being also more stable than SEQ ID NO: 41, underlined by the results obtained with SEQ ID NO: 9.

Example 4: Enzyme performance in laundry applications (stain removal)

Wash performance was measured for the hybrid amylases on cotton soiled with starch (stain types EMPA161 and CS28) purchased from Swissatest Testmaterilien AG and CFT (Center for Testmaterials B.V.). The amylase according to SEQ ID NO: 39 is used as reference for the other amylases being hybrid molecules. SEQ ID NO: 54 is for example the hybrid of SEQ ID NO: 42 and SEQ ID NO: 44 containing a double deletion at the positions 183 and 184 and further mutations at the positions 430 and 454. Also other amylases chosen (SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO: 27) are hybrids with significant changed C-domains over SEQ ID NO: 54.

Amylases were dosed at 0.05, 0.1, 0.2, or 0.4 ppm in 2.5 mM hard water (14 °dH, deutsche Harte) plus 3.3 g/L detergent ESI (Maranil DBS/LC LAS 5,5% w/w, Edenor coco fatty acid C12-C18 coco fatty acid 2,4% w/w, Lutensol A07 AEO 5,4% w/w, Texapon N70 FAEO 5,4% w/w, 1,2 propylene glycol 6,0% w/w, ethanol 2,0% w/w, KOH 2,2% w/w) plus 3% sodium citrate resulting in an pH of 8.0-8.5. Wash was conducted by means of a Launder-O-meter (SDS Atlas) for 30 minutes at 40°C. Wash liquor was removed then the fabric was rinsed three times with water. The samples were dried overnight at room temperature. Wash performance was measured using digital image analysis of washed stains by means of a spectrophotometer (ELREPHPO, Datacolor; the average L* AB intensity was normalized to the detergent base without amylase; and then average of the four data points (0.05, 0.1, 0.2, or 0.4 ppm) is provided.

Wash performance on test stains CS 28 and EMPA 161 is shown as ddE to show the amylase specific effects (dE amy - dE detergent base). ddE

ddE

The results, as provided in the tables above, show that the hybrid amylase enzymes have an improved performance (stain removal) when compared to the parent enzyme SEQ ID NO: 39.

Example 5: Stability testing by heat challenge

Hybrid enzymes of the invention can be stabilized by further changes in the amino acids sequences. This was tested by storing the variants at elevated temperature and measuring the residual activity after certain time.

Herefore, the enzymes were diluted to ~20 pg/mL into 0.1 M Hepes pH 8.0 and challenged at 92°C for 10 min, then chilled at 4°C using a PCR machine. Unchallenged controls were kept at 4°C. To measure residual activity, challenged enzymes and controls were diluted 10 fold into 1% Red Starch prepared in 0.1 M Hepes pH 8.0 and 0.05% Tween20 following the manufacturer's instructions (Megazyme) and incubated at room temperature for 10 min. Reaction was quenched with two volumes of ice-cold ethanol, incubated for 10 min, then spun down. Supernatant was transferred into new plates and absorbance was read at 510 nm. Improvement Factor (IF) was calculated as the ratio between the specific variant and the parent SEQ ID NO: 54.

The mutations are introduced in SEQ ID NO: 54 using the numbering according to SEQ ID NO:

39.

Example 6: Storage of hybrid variants in liquid Laundry detergent The stabilization of hybrid enzymes of the invention by further changes in the amino acids sequences was further tested by storing the variants in liquid detergent and measuring the residual activity after certain time.

Storage in liquid laundry detergent was done by incubating the amylases in a ES1-C detergent solution (Maranil DBS/LC LAS 5,5% w/w, Edenor C12-C18 coco fatty acid 2,4% w/w, Lutensol A07 AEO 5,5% w/w, Texapon N70 FAEO 5,5% w/w, 1,2 propylene glycol 6,0% w/w, ethanol 2,0% w/w, KOH 2,2% w/w and Na-citrate 3%, pH 8) including 1% of Bacillus lentus alkaline protease (BLAP) with R101E mutation and stored at 37°C for 7 days. Samples were taken out at day 0 and 7 and diluted in 50mMMOPS, pH7 before being analyzed with Infinity amylase reagent at room temperature. The activity is the slope at 405nm calculated as the 5 point MaxV over 5 minutes. The improvement factor (IF) over the reference hybrid amylase SEQ ID NO: 54 is calculated by taking percent residual activity after storage of each variant and dividing it by the percent residual activity after storage of Seq ID NO: 54. The mutations are introduced in SEQ ID NO: 54 using the numbering according to SEQ ID NO: 39.

Example 7: Microscale wash tests for hybrid variants Hybrids of the invention can be made more efficient in cleaning performance by minor changes in the amino acids sequences. This was tested by microscale wash trials. Variants were used to wash aged corn starch stains (CS-126) in 3.3 g/L ES1_C (Lutensit A-LBS LAS 5,5% w/w, Edenor coco fatty acid C12-C18 coco fatty acid 2,4% w/w, Lutensol A07 AEO 5,5% w/w, Texapon N70 FAEO 5,5% w/w, 1,2 propylene glycol 6,0% w/w, ethanol 2,0% w/w, KOH 2,2% w/w and Na-citrate 3%, pH 8) with 2,78 mM hard water (15.5 °dH, deutsche Harte) at room temperature at 0.02, 0.05 and 0.1 ppm of amylase added to the wash for 60 minutes before being rinsed for 5 min under running water, dried and measured for intensity of the reflected light as an average of the RGB values. A ratio of the performance of specific variants vs. that of SEQ ID NO: 54 was made and a number above 1 indicates higher performance.

The mutations are introduced in SEQ ID NO: 54 using the numbering according to SEQ ID NO: 39.

Claims

CLAIMS The invention claimed is:

1. An isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44.

2. The polypeptide according to claim 1 having alpha-amylase activity consisting of an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44.

3. The polypeptide according to claim 1 or 2, wherein the A and B domain has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the A and B domain having the amino acid sequence of SEQ ID NO: 42.

4. The polypeptide according to any of the preceding claims, wherein the C domain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the C domain having the amino acid sequence of SEQ ID NO: 44.

5. The polypeptide according to any of the preceding claims comprising a substitution, deletion, and/or insertion at one or more positions.

6. The polypeptide according to any of the preceding claims comprising the sequence

TQXD YLDHPDVIGWTREGDXXHXXS GLAXLMSDGPXGXKWMXV GKNNAGEXWXDIT GNQTNT VTINXD GXGQFXVXXGSXSI YXQX, wherein X can be any ammo acid.

7. The polypeptide according to any of the preceding claims wherein the polypeptide has one or more amino acid residues selected from the group consisting of 402R,H; 419S,G,D; 420V, I; 422, A, V;

423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V, I; 452Y,H,R; 466K,R;

469W,S; 473H,Q,R; 475S,N; 476G,E; 479V, A; 483V, I; and 485R,Q,K according to the numbering of SEQ ID NO: 39.

8. The polypeptide according to any of the preceding claims comprising a substitution at one or more positions selected from the group consisting of 430 and 454, preferably selected from the group consisting of I430M and M454I, according to the numbering of SEQ ID NO: 39.

9. The polypeptide according to any of the preceding claims comprising an amino acid residue at one or more of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 432, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39.

10. The polypeptide according to any of the preceding claims comprising an amino acid residue at one or more of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40.

11. The polypeptide according to any of the preceding claims comprising a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

12. The polypeptide according to any of the preceding claims comprising a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO: 39, preferably one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E/N/Q/S and E190P according to the numbering of SEQ ID NO: 39.

13. The polypeptide according to any of the preceding claims comprising:

(a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37;

(b) an amino acid sequence encoded by a polynucleotide having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38,

(I) a coding sequence of SEQ ID NO:54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or (h) a polynucleotide shown in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; or

(d) a fragment of (a), (b), or (c) having amylase activity.

14. The polypeptide according to any of the preceding claims, wherein the amylase has an increase in expression, activity, thermostability, stability, performance in laundry, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, or any combination thereof compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40, preferably, the amylase has an increase in thermostability compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO: 40.

15. An isolated, a synthetic, or a recombinant nucleic acid comprising:

(a) a nucleic acid sequence having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQID NO:34, SEQID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity;

(b) a nucleic acid sequence encoding a polypeptide having at least at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least, 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity, or encoding a polypeptide of any of the preceding claims;

(c) a polynucleotide that hybridizes under high stringency conditions with the complement of (I) a coding sequence of SEQ ID NO:54, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or

(h) a polynucleotide shown in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38;

(e) a nucleic acid sequence fully complementary to any of (a) to (d).

16. A nucleic acid construct comprising the polynucleotide of claim 15.

17. An expression vector comprising the polynucleotide of claim 15 or the nucleic acid construct of claim 16.

18. A host cell comprising the polynucleotide of claim 15, the nucleic acid construct of claim 16, or the expression vector of claim 17.

19. A composition comprising the isolated, synthetic, or recombinant polypeptide having alpha- amylase activity according to any of claims 1-14.

20. The composition of claim 19, further comprising at least one second enzyme selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a mannanase, a pectinase, xylanase, and a nuclease.

21. A method of making the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity according to any of claims 1-14, comprising: providing a nucleic acid sequence encoding the polypeptide, transforming the nucleic acid sequence into an expression host, cultivating the expression host to produce the polypeptide, and optionally purifying the polypeptide.

22. A method of preparing a dough or a baked product prepared from the dough, the method comprising adding the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity according to any of claims 1-14 to the dough and baking it.

23. A method of use of the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity according to any of claims 1-14, for cleaning or washing textiles, hard surfaces, or dishes, for processing starch, for making ethanol, for processing pulp or paper, or for feeding an animal, preferably for cleaning or washing textiles, hard surfaces, or dishes.

24. A method of making an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising the step of making a hybrid from at least two different amylases, wherein the hybrid comprises an A and B domain and a C domain and wherein the amino acid sequence of the A and B domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44.

25. A method of use of a C domain of a first amylase said C domain having an amino acid sequence which has at least 75% identity to the amino acid sequence of SEQ ID NO: 44 for improving one or more properties selected from the group consisting of stability, pH profile, expression, activity, thermostability, specific activity, substrate specificity, pH-dependent activity, pH- dependent stability, oxidative stability, Ca2+ dependency, performance in laundry, processing starch, cleaning textiles, cleaning hard surfaces, cleaning dishes, making ethanol, processing pulp or paper, and feeding an animal of a second alpha amylase having an A and B domain with at least 75 % identity to the amino acid sequence of SEQ ID NO: 42 said use comprising replacing the C domain of the second alpha-amylase with the C domain of the first alpha- amylase.