Α-amylase mutant, and preparation method therefor and use thereof

Abstract

Provided are an α-amylase mutant having an improved enzyme activity, thermal stability, and / or low-pH stability, a polynucleotide encoding the α-amylase mutant, a recombinant expression vector containing the polynucleotide, a host cell expressing the α-amylase mutant, a composition containing the α-amylase mutant, and a preparation method therefor and the use thereof.

Description

α-Amylase mutants, their preparation methods and applications

[0001] Cross-citation of related applications

[0002] This application claims priority to Chinese patent application CN 202411997994.9, filed on December 31, 2024, which is incorporated herein by reference in its entirety. Technical Field

[0003] This application relates to the field of protein engineering technology, specifically to α-amylase mutants, their preparation methods, and their applications. Background Technology

[0004] α-Amylase (α-1,4-glucan-4-glucan hydrolase, EC 3.2.1.1) hydrolyzes the α-1,4-glycosidic bonds in starch molecules, thus breaking down starch into dextrins, oligosaccharides, and monosaccharides. α-Amylase is one of the earliest, most widely used, and highest-volume enzyme preparations in industry, accounting for approximately 25% of the global enzyme market. In food processing, α-amylase is used in the initial stage of starch processing (liquefaction); in home care, it is used as a detergent base; in the textile industry, it is used for paper desizing; and in the pharmaceutical field, it is used to produce drug carriers and drug prodrugs.

[0005] In various industries such as starch sugar, citric acid, and alcohol, starch is first liquefied by amylase and then saccharified by saccharifying enzymes to produce glucose for subsequent production. However, amylase is easily inactivated above 100°C or below pH 5.0, while the jet liquefaction temperature in the starch liquefaction process can reach above 110°C, and the optimal pH for the subsequent saccharification step is around 4.5. Therefore, the starch sugar industry requires α-amylases with improved enzyme activity, thermal stability, and / or low pH stability.

[0006] α-Amylases contain three distinct domains, A, B, and C (see Machius et al., 1995, J. Mol. Biol. 246:545-559). Domain A is the predominant catalytic domain, typically consisting of a (β / α)8-barrel structure (TIM barrel). Domain A contains several key amino acid residues essential for catalytic activity, such as aspartic acid, glutamic acid, and histidine. These residues play a crucial role in the catalytic reaction, stabilizing the substrate transition state through hydrogen bonding and electrostatic interactions. Domain B is usually a loop structure inserted into domain A, sometimes referred to as the insertion domain. This domain can influence the enzyme's substrate specificity and stability. The size and shape of domain B may vary in α-amylases from different sources. Domain C is typically located at the C-terminus and has a β-sheet structure. This domain plays a supporting role in maintaining the overall stability and proper folding of the enzyme.

[0007] Current research on α-amylases includes isolating new amylases with superior performance from wild types, obtaining mutants with even better performance by mutating known or newly isolated amylases, and obtaining heterozygous amylases by hybridizing different regions of amylases from different sources. However, no research has yet been published on the functional regions within the amylase structural domains. Summary of the Invention

[0008] This invention provides α-amylase mutants, their preparation methods, and applications. The α-amylase mutants exhibit enhanced enzyme activity, thermostability, and / or low pH stability, particularly improved stability under high temperature and low pH conditions compared to parental α-amylase, wild-type α-amylase, or other α-amylase mutants. This invention also provides a method for improving the enzyme activity, thermostability, and / or low pH stability of α-amylase, comprising, based on the three-dimensional structure of α-amylase, [the following steps are taken] around region 3, region 4, region 6, and / or region 6. The present invention modifies the amino acid composition of the α-amylase within a specified range, and prepares α-amylase mutants by the method. Based on these improved properties, the α-amylase mutants of the present invention can produce better results in industrial production, such as being more suitable for liquefaction reactions in the starch industry and improving liquefaction efficiency. Attached Figure Description

[0009] Figure 1 shows the three-dimensional structural comparison results of amylase (M35, SEQ ID NO:26) from Bacillus licheniformis and amylase (SEQ ID NO:5) from Bacillus thermophilus. Detailed Implementation

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0011] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples described herein are illustrative only and not intended to be restrictive.

[0012] When the terms “about” and “approximately” are used with numerical variables, they generally mean that the value of the variable and all values ​​of the variable are within the measurement or experimental error (e.g., the 95% confidence interval of the mean) or within a wider range of specified values ​​(e.g., ±5% or ±10%).

[0013] The term "comprising," or its variations such as "containing," "having," or "including," means to include the stated steps or elements, but does not exclude any other steps or elements. "Constitutes of," means excluding steps or elements not listed. "Substantially constitutes of," means not excluding steps or elements that do not substantially affect the fundamental and novel features of the protected invention. The term "comprising" and its variations also include the cases of "consisting of specific steps or elements" and "substantially constitutes specific steps or elements."

[0014] When referring to a numerical range, it should be understood that the specific values ​​of its upper and lower limits are disclosed, as well as all intermediate ranges included therein, such as the intermediate range between its upper or lower limit and any intermediate value, or the intermediate range between any two intermediate values. Furthermore, any intermediate ranges, subranges, and all individual numerical values ​​described in the numerical range can be excluded from the numerical range.

[0015] The term “and / or” should be understood as any one of the multiple elements connected by the term, or a combination of any number of elements.

[0016] The term "one or more" as used in this invention may include, for example, 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 or more.

[0017] The term "three-dimensional structure" in this invention is used interchangeably with "protein structure," "spatial structure," "3D structure," or "tertiary structure," referring to the spatial structure of a protein molecule (e.g., the α-amylase or its mutant described herein). Tertiary structure refers to the three-dimensional spatial structure formed by further coiling or folding of the polypeptide chain of a protein on top of its secondary structures, such as α-helices, β-sheets, turns, random coils, and loops. Those skilled in the art are familiar with methods for determining the three-dimensional structure of proteins, such as, but not limited to, X-ray diffraction, nuclear magnetic resonance, and cryo-electron microscopy. The three-dimensional structure of proteins can also be predicted using computational biology methods, such as algorithms, software, and / or programs, when the primary structure of the protein, i.e., the amino acid sequence, is known. Such techniques include, but are not limited to, RGN (Recurrent Geometric Network), NEMO (Neural Energy Modeling and Optimization), AlphaFold (e.g., AlphaFold2 or AlphaFold3), and EMSFold.

[0018] The terms "regions overlapping in three-dimensional structures" or "amino acids overlapping in three-dimensional structures" refer to the spatially overlapping regions or amino acids on two proteins when their three-dimensional structures are compared to maximize overlap (e.g., by rotation, translation, etc.). In this disclosure, these may be referred to as "corresponding regions" or "corresponding amino acids." Those skilled in the art can obtain information about the overlapping (or corresponding) regions or amino acids in the three-dimensional structures of two proteins using software or databases known in the art (such as those mentioned above).

[0019] The term "distance" or "spatial distance" between amino acids refers to the spatial distance between two specific amino acids in a protein molecule. Specifically, this distance refers to the distance between specific atoms (usually α-carbon atoms or side-chain atoms) of the two amino acids, typically expressed in angstroms (Å). Measurements are taken in units of angstrom, where 1 angstrom equals 0.1 nanometer (nm). That is, 0.4 nanometers.

[0020] The term "root mean square deviation" (RMSD) is used in structural biology to compare the similarity of molecular structures. A smaller RMSD value indicates greater similarity between two structures. It is generally believed that... At this point, the two structures have a high degree of similarity. The formula for calculating RMSD is as follows:

[0021] Where: N is the number of paired atoms; δi is the position of the i-th atom in a certain frame minus its position in the reference conformation (position offset).

[0022] The formula can be broken down into the following steps: For each pair of corresponding atoms, calculate their coordinate difference; calculate the square of the coordinate difference for each pair of atoms; sum the squares of the differences between all paired atoms; divide the sum by the number of paired atoms (N). Root the average value to obtain the RMSD value.

[0023] The term "amino acid alteration" refers to the insertion, deletion, and / or substitution of one or more amino acids. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; insertion means adding one or more (e.g., 1-5) amino acids adjacent to an amino acid occupying a position.

[0024] The term "mutant" or "variant" refers to a polypeptide that has one or more amino acid insertions, deletions, and / or substitutions relative to a parent polypeptide. A mutant retains at least one activity of the parent polypeptide (e.g., α-amylase activity), but may have variations in activity levels; for example, a mutant may remain unchanged or improve upon at least one activity or property relative to the parent polypeptide. The term "functional variant" refers to a mutant that retains at least one activity of the parent polypeptide (e.g., α-amylase activity).

[0025] The term "parent" refers to a polypeptide that has undergone amino acid alterations to produce a mutant; the term can also refer to a polypeptide to which the mutant of the present invention is compared. The parent can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source should mean that a parent encoded by a polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, the parent is extracellularly secreted.

[0026] The term “α-amylase” is synonymous with the term “polypeptide with α-amylase activity.” The term “α-amylase activity” or “amylase activity” refers to the activity of hydrolyzing the α-1,4-glycosidic bonds in starch molecules to produce dextrins, oligosaccharides, and monosaccharides. Therefore, as used herein, the term “α-amylase” refers to an enzyme (enzyme class; EC 3.2.1.1) with α-amylase activity that hydrolyzes the α-bonds of large α-linked polysaccharides (e.g., starch and glycogen) to produce oligosaccharides, glucose, and maltose. The terms “α-amylase,” “α-amylase,” and “amylase” are used interchangeably and have the same meaning and purpose herein.

[0027] The term "stability" refers to the property of α-amylase or its mutants to maintain a certain level of enzyme activity under specific environmental conditions during storage, use, or handling. For example, "thermal stability" or "stability at high temperatures" refers to the ability of α-amylase or its mutants to maintain a certain level of enzyme activity after a period of time at a specific or higher temperature; this is also referred to as "heat resistance" in this document. "Low pH stability" refers to the ability of α-amylase or its mutants to maintain a certain level of enzyme activity after a period of time at a lower pH; this is also referred to as "acid resistance" in this document. When comparing aspects such as stability, "improved stability" refers to the higher residual enzyme activity of α-amylase or its mutants under specific conditions (e.g., after a period of time at high temperatures and / or low pH) compared to other α-amylase mutants and / or parental α-amylases and / or wild-type α-amylases. Improved stability of α-amylase or its mutants at high temperatures is also referred to as improved heat resistance in this document. Improved stability of α-amylase or its mutants at low pH is also referred to as improved acid resistance in this document.

[0028] The term "residual enzyme activity %" refers to the percentage of enzyme activity of α-amylase or its mutant under specific conditions (e.g., after a period of time at a specific temperature and / or a specific pH, preferably after a period of time at a high temperature and / or a low pH) relative to the enzyme activity of the untreated enzyme, wherein the enzyme activity before and after treatment is determined by the same method.

[0029] The term "wild-type" enzyme refers to an enzyme expressed by a naturally occurring organism or cell (such as bacteria or fungi). "Naturally occurring" means without artificial mutagenesis or genetic manipulation.

[0030] The term "recombinant" when used to refer to cells, nucleic acids, proteins, or vectors indicates that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells that have been modified in this way. Therefore, recombinant cells express genes that are not present in the natural (non-recombinant) form of the cell; or express natural genes that are otherwise abnormally, insufficiently, or not expressed at all.

[0031] The term "heterogeneous" refers to nucleic acids or peptides that are artificially introduced into cells and / or are not naturally present in the cells in which they exist.

[0032] The term “expression” in the context of this invention includes any step involved in the generation of the α-amylase mutant of this invention, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0033] The term “expression vector” is defined herein as a linear or circular DNA molecule containing a polynucleotide encoding a protein such as the α-amylase mutant of the present invention, and said polynucleotide being operatively linked to additional nucleotides (e.g., control sequences) provided for its expression.

[0034] The term "host cell" includes cells transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing polynucleotides encoding α-amylase mutants, and daughter cells of parent cells that are different from parent cells due to mutations during replication, and cells expressing the α-amylase mutant of the present invention.

[0035] The term "control sequence" refers to the nucleotide sequence necessary for the expression of the polynucleotide encoding the α-amylase mutant of the present invention. Each control sequence may be homologous (i.e., from the same gene) or heterologous (i.e., from different genes) to the polynucleotide encoding the α-amylase mutant. These control sequences include (but are not limited to) leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. In some embodiments, the control sequences include at least a promoter and transcription and translation termination signals.

[0036] The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. Mature polypeptides, for example, may undergo N-terminal processing without containing a signal peptide.

[0037] The term "signal peptide" refers to a peptide that is linked to the amino terminus of a mature polypeptide in a read-frame manner and guides the encoded polypeptide into the cell's secretory pathway.

[0038] The term "sequence consistency" describes the correlation between two amino acid sequences or two nucleotide sequences. Sequence consistency refers to the percentage of sequence similarity determined by performing optimal alignment (maximum sequence consistency) of two sequences within a comparison window. Optimal alignment can be achieved through additions or deletions (i.e., gaps). The sequence consistency percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs under optimal alignment mode to generate the number of matching positions, dividing the number of matching positions by the total number of positions compared, and then multiplying the result by 100 to obtain the sequence consistency percentage. Sequence consistency between two amino acid sequences can be determined using available local alignment tools (e.g., BLAST) or global alignment tools (e.g., the Needleman-Wunsch algorithm). For example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) can be used to determine sequence identity between two amino acid sequences. The parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). Alternatively, the parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL substitution matrix (the EMBOSS version of NCBI NUC4.4).

[0039] The term "corresponding to a position" in particular, when used to describe an amino acid position in the primary structure of a protein (i.e., the amino acid sequence), refers to the position in the reference amino acid sequence that corresponds to a specific position in the reference amino acid sequence when the queried amino acid sequence is best aligned with a reference amino acid sequence (e.g., the best alignment as described above). The identification of corresponding amino acid residues in another α-amylase can be determined by comparing multiple peptide sequences using several computer programs with their respective default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparison by logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acids Research, 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research, 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research, 33:511-518; Katoh and Toh, 2007, Bioinformatics, 23:372-374; Katoh et al., 2009, Methods in Molecular Biology, 537:39-64; Katoh and Toh, 2010, Bioinformatics, 26:1899-1900) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research, 22:4673-4680).

[0040] When α-amylase deviates from the reference amino acid sequence of a polypeptide, rendering traditional sequence-based comparisons ineffective (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), alternative pairwise sequence comparison algorithms can be used. Higher sensitivity in sequence-based searches can be achieved using search programs that utilize the probabilistic representation (spectrum) of polypeptide families to search a database. For example, the PSI-BLAST program generates multiple spectra through an iterative database search process and can detect distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even higher sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J.Mol.Biol.287:797-815; McGuffin and Jones, 2003, Bioinformatics.19:874-881) utilize information from multiple sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J.Mol.Biol.313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose. For proteins with known structures, several tools and resources are available for retrieving and generating structural alignments. For example, SCOP superfamily structures of proteins have already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms, such as distance alignment matrix (Holm and Sander, 1998, Proteins. 33: 88-96) or combined extension (Shindyalov and Bourne, 1998, Protein Engineering. 11: 739-747), can be used to align two or more protein structures, and implementations of these algorithms can also be used to query structure databases with the target structure in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics. 16: 566-567).

[0041] The mutant representation method of this invention: Amino acids are represented by conventional single-letter or three-letter names. As is known to those skilled in the art, the single-letter names for amino acids are as follows: A for alanine, corresponding to the three-letter name Ala; C for cysteine, corresponding to the three-letter name Cys; D for aspartic acid, corresponding to the three-letter name Asp; E for glutamic acid, corresponding to the three-letter name Glu; F for phenylalanine, corresponding to the three-letter name Phe; G for glycine, corresponding to the three-letter name Gly; H for histidine, corresponding to the three-letter name His; I for isoleucine, corresponding to the three-letter name Ile; K for lysine, corresponding to the three-letter name Lys; L for leucine, ... The three letters corresponding to these three letters are named Leu; M is methionine, corresponding to Met; N is asparagine, corresponding to Asn; P is proline, corresponding to Pro; Q is glutamine, corresponding to Gln; R is arginine, corresponding to Arg; S is serine, corresponding to Ser; T is threonine, corresponding to Thr; V is valine, corresponding to Val; W is tryptophan, corresponding to Trp; and Y is tyrosine, corresponding to Tyr.

[0042] In this paper, the following nomenclature is used to represent amino acid insertions: original amino acid, position, original amino acid, inserted amino acid. For example, D18DA can be used to represent an amino acid insertion, where 18 indicates that an amino acid insertion occurs after position 18 of the reference sequence, and amino acid A is inserted. In this paper, the following nomenclature is used to represent amino acid deletions: original amino acid, position, *. For example, "I181*" can be used to represent an amino acid deletion, where 181 indicates that a deletion occurs at position 181 of the reference sequence, losing the amino acid I that originally occupied that position. The original amino acid preceding the position can be omitted to indicate any amino acid deletion at a specific position; for example, "181*" can be used to represent any amino acid deletion at position 181. In this paper, the following nomenclature is used to represent amino acid substitutions: original amino acid, position, existing amino acid. For example, "V59A" can be used to represent an amino acid substitution, where 59 indicates that a substitution occurs at position 59 of the reference sequence, the V preceding 59 is the amino acid that occupied that position before the substitution, and the A following 59 is the amino acid that occupied that position after the substitution. When an amino acid change is represented by the form "V3(AAPF)", it means that the amino acid at that position was V before the change, and AAPF after the change. The original amino acid at the beginning of the position can be omitted, indicating that any amino acid at a specific position is replaced by a specific amino acid, or that a specific amino acid is present at a specific position. For example, "59A" can represent that any amino acid at position 59 is replaced by A, or that amino acid A is present at position 59. Similarly, "3(AAPF)" can represent that any amino acid at position 3 is replaced by AAPF (SEQ ID NO: 65), or that AAPF (SEQ ID NO: 65) is present at position 3. Multiple mutations are connected by " / ", for example, "V59A / N122D / S124N / Q128K" means that amino acid V at position 59 is replaced by A, amino acid N at position 122 is replaced by D, amino acid S at position 124 is replaced by N, and amino acid Q at position 128 is replaced by K. For example, "59A / 122D / 124N / 128K" means that any amino acid at position 59 is replaced with A, any amino acid at position 122 is replaced with D, any amino acid at position 124 is replaced with N, and any amino acid at position 128 is replaced with K; or the amino acid at position 59 is A, the amino acid at position 122 is D, the amino acid at position 124 is N, and the amino acid at position 128 is K.

[0043] It should be understood that in this invention, when referring to amino acid changes in certain regions or positions of α-amylase, or when an α-amylase mutant contains amino acid changes in certain regions or positions, changes in amino acids at other positions are not excluded. For any position not mentioned, the amino acids at that position may or may not be changed relative to the parental sequence. In some embodiments, the α-amylase mutant contains only the listed amino acid changes relative to the parental sequence and does not contain any amino acid changes at unlisted positions.

[0044] It should be understood that in this invention, when referring to a specific positional amino acid change in α-amylase or an α-amylase mutant containing a specific positional amino acid change, it can be understood as the mutant containing the described mutated amino acid at said position. Considering the possibility of using different parental α-amylases, when the parental α-amylase already possesses the described mutated amino acid at that position (e.g., the parental α-amylase is a mutant of wild-type α-amylase and has the described mutated amino acid at that position relative to the wild-type α-amylase), referring to a specific positional amino acid change in α-amylase or an α-amylase mutant containing that positional amino acid change also includes the case where the mutant has no change in the corresponding amino acid at that position relative to the parental α-amylase; when the parental α-amylase has different amino acids at that position, referring to a specific positional amino acid change in α-amylase or an α-amylase mutant containing that position also includes the case where the different amino acid is mutated into the mutated amino acid.

[0045] Unless otherwise stated, nucleic acids are written from left to right in the 5' to 3' direction in this document, and amino acid sequences are written from left to right in the direction from the amino terminus to the carboxyl terminus.

[0046] Through research, the inventors discovered that amino acid alterations in specific regions of the three-dimensional structure of α-amylase from Bacillus stearothermophilus can improve the enzyme activity, thermal stability, and / or low pH stability of α-amylase. Furthermore, they found that amino acid alterations in corresponding regions of other α-amylases with similar three-dimensional structures can also improve the enzyme activity, thermal stability, and / or low pH stability of α-amylase.

[0047] Therefore, this invention provides a method for preparing α-amylase with improved enzyme activity, thermal stability, and / or low pH stability (or a method for modifying α-amylase to improve its enzyme activity, thermal stability, and / or low pH stability), comprising altering amino acids in specific regions of an α-amylase having a specific three-dimensional structure (specifically described below), such alterations improving the enzyme activity, thermal stability, and / or low pH stability of the α-amylase. Methods for altering amino acids in α-amylase are well known to those skilled in the art, such as site-directed mutagenesis and homologous recombination.

[0048] The present invention also relates to a method for preparing α-amylase mutants with improved enzyme activity, thermal stability and / or low pH stability by the above method, and the α-amylase mutants obtained by the method.

[0049] This invention also relates to α-amylase mutants possessing α-amylase activity, thermal stability, and / or low pH stability, particularly stability at high temperatures and low pH. In some embodiments, the α-amylase mutants of this invention exhibit increased α-amylase activity, and / or increased thermal stability, and / or increased low pH stability compared to parental α-amylase and / or wild-type α-amylase and / or other α-amylase mutants. In some embodiments, the α-amylase mutants have amino acid alterations (e.g., relative to parental α-amylase) in the specific regions, preferably these amino acid alterations enhancing the enzyme activity, thermal stability, and / or low pH stability of the α-amylase. The properties of the α-amylase mutants of this invention make them particularly useful in industrial production and can be widely applied in fields such as food, home care, textiles, feed, or medicine, specifically including but not limited to starch liquefaction, saccharification, fermentation, brewing, baking, textile desizing, textile washing, and increasing digestibility in animal feed.

[0050] The term "high temperature" refers to at least about 80°C, at least about 85°C, at least about 90°C, at least about 95°C, or at least about 100°C, for example, 80°C-120°C, preferably 85°C-115°C, and more preferably 95°C-110°C. The term "low pH" refers to a pH value of 4.0-5.8, for example, 4.0-5.0, preferably 4.0-4.5, and more preferably 4.0-4.2.

[0051] In some embodiments, the α-amylase activity of the α-amylase mutant of the present invention is 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 90%, at least 95%, or at least 100% higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutants, for example, but not limited to, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. The enzyme activity can be detected, for example, by an iodine-starch colorimetric method, whereby the α-amylase is reacted with soluble starch, followed by the addition of iodine solution, and the enzyme activity is determined by detecting the absorbance at a specific wavelength (e.g., 660 nm) and comparing it to a control. In some embodiments, the enzyme activity assay is performed at 70°C and pH 6.0. In some embodiments, the enzyme activity assay is performed after maintaining the temperature at 100°C and pH 4.0 for 30 minutes.

[0052] In some embodiments, the residual enzyme activity or tolerance factor of the α-amylase mutant of the present invention under high-temperature conditions, particularly above 80°C (e.g., 80°C-120°C, 80°C-115°C, e.g., 85°C-110°C, e.g., about 100°C), is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity or tolerance factor of the α-amylase mutant of the present invention at 100°C is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity or tolerance factor of the α-amylase mutant of the present invention after being held at 100°C for 30 minutes is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at 70°C.

[0053] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C for 30 minutes to the residual enzyme activity % of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same treatment is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, or greater than 3. 30, greater than 3.40, greater than 3.50, greater than 3.60, greater than 3.70, greater than 3.80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, about 1.05, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00.

[0054] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C for 30 minutes to the residual enzyme activity % of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant under the same treatment, i.e., the tolerance factor of the α-amylase mutant of the present invention is greater than 1.00, greater than 1.25, greater than 1.50, greater than 1.75, greater than 2.00, greater than 2.25, greater than 2.50, greater than 2.75, greater than 3.00, greater than 3.25, greater than 3.50, greater than 3.75, greater than 4.00, greater than 4.25, greater than 4.50, greater than 4.75, greater than 5.00, greater than 5.25, greater than 5.50, greater than 5.75, greater than 6.00, greater than 6.25, greater than 6.50, greater than 6.75, greater than 7.00, greater than 7.25, greater than 7.50, greater than 7.75, greater than 8.00, greater than 8.2 5. Values ​​greater than 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, or 10.50 or higher, such as, but not limited to, approximately 1.25, approximately 1.50, approximately 1.75, approximately 2.00, approximately 2.25, approximately 2.50, approximately 2.75, approximately 3.00, approximately 3.25, approximately 3.50, approximately 3.75, approximately 4.00, or approximately 4.2. 5. Approximately 4.50, Approximately 4.75, Approximately 5.00, Approximately 5.25, Approximately 5.50, Approximately 5.75, Approximately 6.00, Approximately 6.25, Approximately 6.50, Approximately 6.75, Approximately 7.00, Approximately 7.25, Approximately 7.50, Approximately 7.75, Approximately 8.00, Approximately 8.25, Approximately 8.50, Approximately 8.75, Approximately 9.00, Approximately 9.25, Approximately 9.50, Approximately 9.75, Approximately 10.00, Approximately 10.25, Approximately 10.50.

[0055] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being held at 100°C for 30 minutes is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at 70°C.

[0056] In some embodiments, the residual enzyme activity percentage of the α-amylase mutant of the present invention under low pH conditions, particularly pH 4.0-5.8 (e.g., 4.0-5.0, e.g., 4.0-4.5, e.g., about 4.0), is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity percentage of the α-amylase mutant of the present invention at pH 4.0 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity percentage of the α-amylase mutant of the present invention after being held at pH 4.0 for 30 minutes is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at pH 6.0.

[0057] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention under low pH conditions, particularly pH 4.0-5.0 (e.g., about 4.5 or about 4.2), is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.5 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.2 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.2 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant at pH 4.5. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at pH 4.5 for 30 minutes is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at pH 4.2 for 30 minutes is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at pH 4.2 for 30 minutes is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant after being kept at pH 4.5 for 30 minutes. In some embodiments, the enzyme activity assay of the untreated mutant is performed at pH 6.0.

[0058] In some embodiments, the α-amylase mutant of the present invention is at pH The ratio of residual enzyme activity % at 4.0 to the residual enzyme activity % of parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same treatment, i.e., the tolerance factor of the α-amylase mutant of the present invention is greater than 1.00, greater than 1.25, greater than 1.50, greater than 1.75, greater than 2.00, greater than 2.25, greater than 2.50, greater than 2.75, greater than 3.00, greater than 3.25, greater than 3.50, greater than 3.75, greater than 4.00, greater than 4.25, greater than 4.50, greater than 4.75, greater than 5.00, greater than 5.25, greater than 5.50, greater than 5.75, greater than 6.00, greater than 6.25, greater than 6.50, greater than 6.75, greater than 7.00, greater than 7.25, greater than 7.50, greater than 7.75, greater than 8.00, greater than 8.25, greater than 8.50, and greater than 8.7. 5. Values ​​greater than 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, or 10.50 or higher, such as, but not limited to, approximately 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, or 4.50. Approximately 4.75, approximately 5.00, approximately 5.25, approximately 5.50, approximately 5.75, approximately 6.00, approximately 6.25, approximately 6.50, approximately 6.75, approximately 7.00, approximately 7.25, approximately 7.50, approximately 7.75, approximately 8.00, approximately 8.25, approximately 8.50, approximately 8.75, approximately 9.00, approximately 9.25, approximately 9.50, approximately 9.75, approximately 10.00, approximately 10.25, approximately 10.50.

[0059] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.0 is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at pH 6.0.

[0060] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.5 or pH 4.2 to the residual enzyme activity % of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same treatment is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, and greater than 3. 50, greater than 3.60, greater than 3.70, greater than 3.80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, about 1.05, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00.

[0061] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.2 to the residual enzyme activity % of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant at pH 4.5 is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, greater than 3.50, greater than 3.60, greater than 3.70, greater than 3. .80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, about 1.05, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00.

[0062] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.5 is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at pH 6.0.

[0063] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.2 is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at pH 6.0.

[0064] In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention under high temperature and low pH conditions, particularly above 80°C (e.g., 80°C-115°C, e.g., 85°C-110°C, e.g., about 100°C) and pH of 4.0-5.8 (e.g., 4.0-5.0, e.g., 4.0-4.5, e.g., about 4.0), is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention at 100°C and pH 4.0 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention after being held at 100°C and pH 4.0 for 30 minutes is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some implementations, the enzyme activity of untreated mutants is measured at 70°C and pH 6.0.

[0065] In some embodiments, the α-amylase mutant of the present invention is subjected to a temperature of 100°C and a pH of [missing information]. 4.0 The ratio of residual enzyme activity % after 30 minutes to the residual enzyme activity % of parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same treatment, i.e., the tolerance factor of the α-amylase mutant of the present invention is greater than 1.00, greater than 1.25, greater than 1.50, greater than 1.75, greater than 2.00, greater than 2.25, greater than 2.50, greater than 2.75, greater than 3.00, greater than 3.25, greater than 3.50, greater than 3.75, greater than 4.00, greater than 4.25, greater than 4.50, greater than 4.75, greater than 5.00, greater than 5.25, greater than 5.50, greater than 5.75, greater than 6.00, greater than 6.25, greater than 6.50, greater than 6.75, greater than 7.00, greater than 7.25, greater than 7.50, greater than 7.75, greater than 8.00, greater than 8.25, greater than 8.50, greater than Values ​​of 8.75, greater than 9.00, greater than 9.25, greater than 9.50, greater than 9.75, greater than 10.00, greater than 10.25, greater than 10.50, or higher, such as, but not limited to, approximately 1.25, approximately 1.50, approximately 1.75, approximately 2.00, approximately 2.25, approximately 2.50, approximately 2.75, approximately 3.00, approximately 3.25, approximately 3.50, approximately 3.75, approximately 4.00, approximately 4.25, approximately 4.5 0, approximately 4.75, approximately 5.00, approximately 5.25, approximately 5.50, approximately 5.75, approximately 6.00, approximately 6.25, approximately 6.50, approximately 6.75, approximately 7.00, approximately 7.25, approximately 7.50, approximately 7.75, approximately 8.00, approximately 8.25, approximately 8.50, approximately 8.75, approximately 9.00, approximately 9.25, approximately 9.50, approximately 9.75, approximately 10.00, approximately 10.25, approximately 10.50.

[0066] In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention after holding at 100°C and pH 4.0 for 30 minutes is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed after holding at 70°C and pH 6.0 for 30 minutes.

[0067] In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention under high temperature and low pH conditions, particularly 80°C-120°C and pH 4.0-5.0 (e.g., 100°C, pH 4.5 or 100°C, pH 4.2), is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention at 100°C and pH 4.5 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention at 100°C and pH 4.2 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at 100°C and pH 4.2 is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutants at 100°C and pH 4.5. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.5 for 30 minutes is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.2 for 30 minutes is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.2 for 30 minutes is higher than that of the residual enzyme activity % of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant after being kept at 100°C and pH 4.5 for 30 minutes. In some embodiments, the parental α-amylase or other α-amylase mutant is mutant LE399 or mutant M35. In some embodiments, the parental α-amylase or other α-amylase mutant is any of the amylases shown in SEQ ID NO:1 or SEQ ID NO:3-14. In some embodiments, the enzyme activity assay of the untreated mutant is performed at 70°C and pH 6.0.

[0068] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C, pH 4.5 or 100°C, pH 4.2 for 30 minutes to the residual enzyme activity % of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same treatment is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, and greater than 1.10, greater than 1.20, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, greater than 1.20, greater than 1.20, greater than 3.30, greater than 3.40, greater than 1.20, greater than 1.20, greater than 1.20, greater than 1.20, greater than 1.20, greater than 1.40, greater than 1.20, greater Values ​​of 3.50, greater than 3.60, greater than 3.70, greater than 3.80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, approximately 1.05, approximately 1.10, approximately 1.20, approximately 1.30, approximately 1.40, approximately 1.50, approximately 1.60, approximately 1.70, approximately 1.80, approximately 1.90, approximately 2.00, approximately 2.10, approximately 2.20, approximately 2.30, approximately 2.40, approximately 2.50, approximately 2.60, approximately 2.70, approximately 2.80, approximately 2.90, approximately 3.00, approximately 3.10, approximately 3.20, approximately 3.30, approximately 3.40, approximately 3.50, approximately 3.60, approximately 3.70, approximately 3.80, approximately 3.90, and approximately 4.00.

[0069] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.2 for 30 minutes to the residual enzyme activity % of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant after being kept at 100°C and pH 4.5 for 30 minutes is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, greater than 3.50, greater than 3.60, greater than 3.70. Greater than 3.80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, about 1.05, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00.

[0070] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.5 for 30 minutes is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at 70°C and pH 6.0.

[0071] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.2 for 30 minutes is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at 70°C and pH 6.0.

[0072] In some embodiments, the α-amylase having a specific three-dimensional structure may be an α-amylase with a similar or even substantially identical three-dimensional structure to the reference *Bacillus stearothermophilus* α-amylase. The α-amylase having this three-dimensional structure can serve as a parent enzyme for which amino acid alterations are performed in the methods of the present invention, or as a parent enzyme for a mutant of the present invention (e.g., the mutant may be comparable to the parent enzyme in terms of mutation site or effect). In some embodiments, the reference *Bacillus stearothermophilus* α-amylase comprises an amino acid sequence as shown in SEQ ID NO: 1 or 5.

[0073] In some embodiments, the three-dimensional structure of any α-amylase or its mutant described in this invention can be determined by any method, algorithm, program, and / or software that can be used to determine, predict, and / or resolve the three-dimensional structure of a protein, such as, but not limited to, X-ray diffraction, nuclear magnetic resonance, cryo-electron microscopy, and computational biology methods. For example, RGN (Recurrent Geometric Network), NEMO (Neural Energy Modeling and Optimization), AlphaFold (e.g., AlphaFold2 or AlphaFold3), EMSFold, etc., can be used to determine or predict the three-dimensional structure of the protein, and the determined or predicted three-dimensional structure can be used in the method of this invention.

[0074] The terms "similar" or "substantially identical" when used for comparing the three-dimensional structures of proteins mean that the three-dimensional structures of two proteins are very similar, or essentially overlap (or coincident), for example, the sizes of the various secondary structures and their relative positions after further folding are basically the same. Various software programs, algorithms, and / or databases are known to be used for comparing the three-dimensional structures of two proteins, including but not limited to PyMol, DaLi, CE (combinational extension), TM-align, SSAP, FLEXPROT, FATCAT, and the PDB database. The use of these software programs, algorithms, and / or databases is well known to those skilled in the art; for example, comparisons can be performed using default parameters.

[0075] The similarity of the three-dimensional structures of two proteins can be assessed using various evaluation metrics known to those skilled in the art, such as the root mean square deviation (RMSD), which represents the average distance difference between all atomic positions of the two structures; a smaller RMSD indicates a closer similarity. Other metrics include the template modeling score (TM-score), which represents the degree of atomic position matching after optimal superlocalization; a larger TM-score indicates greater similarity. The global distance test total score (GDT-TS) represents the proportion of atomic position matching between the two structures at different distance thresholds; a larger GDT-TS indicates greater similarity. Other metrics include Q-score, CAD-score, and MaxSub. These metrics can be obtained using various known software and databases, such as those mentioned above. In some implementations, when comparing the three-dimensional structures of two proteins, an RMSD of less than [a certain value] is considered acceptable. (e.g., less than) Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than Less than or less When the two proteins are identical, their three-dimensional structures can be considered to be basically the same.

[0076] In some embodiments, the three-dimensional structure can be a complete three-dimensional structure, i.e., the three-dimensional structure of the full-length amino acid sequence of the protein; or a partial three-dimensional structure, i.e., the three-dimensional structure of a portion of the amino acid sequence of the protein. In some embodiments, the complete three-dimensional structure of the α-amylase described in this disclosure is compared with the complete three-dimensional structure of a reference enzyme (such as the amylase shown in SEQ ID NO: 1 or 5).

[0077] α-Amylases with similar three-dimensional structures to the reference *Bacillus stearothermophilus* α-amylase can also be considered parental enzymes in this study. These can be naturally occurring (wild-type) polypeptides or mutants prepared by any suitable means. For example, a parental enzyme can be a mutant of a naturally occurring polypeptide whose amino acid sequence has been modified or altered. Therefore, parental α-amylases may have amino acid alterations relative to wild-type α-amylases, such as substitutions, deletions, and / or insertions of one or more amino acids. In some embodiments, the parent enzyme (or an α-amylase with a similar three-dimensional structure to the reference thermophilic Bacillus stearothermophilus α-amylase) may include bacterial α-amylases, such as α-amylases from Gram-positive bacteria, including Bacillus, Clostridium, Exiguobacterium, Enterococcus, Geobacillus, Paenibacillus, Alicyclobacillus, Lactobacillus, Lactococcus, and Oceanobacillium. α-Amylases of bacteria such as *Staphylococcus*, *Streptococcus*, or *Streptomyces*, or α-amylases of Gram-negative bacteria such as those derived from *Campylobacter*, *E. coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Ilyobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, or *Ureaplasma*, or their functional variants thereof.In some embodiments, the parent enzyme (or an α-amylase with a similar three-dimensional structure to the reference *Geobacillus stearothermophilus* α-amylase) may be an α-amylase from *Geobacillus stearothermophilus*, *Bacillus licheniformis*, *Bacillus subtilis*, *Bacillus halmapalus*, *Exiguobacterium acetylicum*, *Exiguobacterium sibiricum*, *Paenibacillus curdlanilyticus*, *Bacillus megaterium*, *Alicyclobacillus Sp. 18711*, or *Bacillus amyloliquefaciens*, or a functional variant thereof.

[0078] The parent can be a naturally occurring (wild-type) polypeptide or a mutant thereof prepared by any suitable means. For example, the parent protein can be a mutant of a naturally occurring polypeptide whose amino acid sequence has been modified or altered. Thus, the parent α-amylase may have one or more amino acid substitutions, deletions, and / or insertions. Thus, the parent α-amylase may be a mutant of the wild-type α-amylase. The term "parent" as used herein can include, for example, wild-type α-amylase of *G. stearothermophilus* (SEQ ID NO:2), and mutants of wild-type α-amylase of *G. stearothermophilus*, such as α-amylases having the amino acid sequences shown in SEQ ID NO:1 or SEQ ID NO:5. SEQ ID NO:1 is obtained by deleting the first to 34th amino acid residues from the N-terminus of wild-type α-amylase (SEQ ID NO:2) and the first to 27th amino acid residues from the C-terminus; SEQ ID NO:5 is obtained by using SEQ ID NO:1 as the parent, deleting the 180th and 181st amino acid residues from the N-terminus, and mutating the 193rd amino acid at the N-terminus to F and the 416th amino acid to G. The term "parent" as used herein can include, for example, the α-amylase shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:9-24, or any α-amylase having at least 60% sequence identity with any polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:9-24. The term "parent" as used herein may also include polypeptides containing any of the fragments in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:9-24.

[0079] In some embodiments, the parent used herein has at least 60% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:9-24, for example, at least 60%, at least 65%, at least 70%, at least 75%, 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 99%, but less than 100%. In some embodiments, the parent is sequence-aligned with the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9-24 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide removed).

[0080] The term "parent" as used herein can include, for example, the mutant LE399 mentioned herein, and can also include mutants containing the V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W mutation, such as mutant M35 described herein. The term "parent" as used herein can include, for example, any of the α-amylases in SEQ ID NO:25-39, or any α-amylase having at least 60% sequence identity with any polypeptide of SEQ ID NO:25-39. The term "parent" as used herein can also include polypeptides containing fragments of any of SEQ ID NO:25-39.

[0081] In some embodiments, the parent used herein has at least 60% sequence identity with the amino acid sequence shown in any of SEQ ID NO:25-39, for example, at least 60%, at least 65%, at least 70%, at least 75%, 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 99%, but less than 100%. In some embodiments, the parent is sequence-aligned with the amino acid sequence shown in any of SEQ ID NO:25-39 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide).

[0082] In some embodiments, the parent used herein may be an α-amylase of the Bacillus genus as shown in SEQ ID NO:44, or a sequence identical to SEQ ID NO:44 with at least 60% sequence identity, such as at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99%, but less than 100%. In some embodiments, the parent is sequence-aligned with the amino acid sequence of SEQ ID NO:44 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide).

[0083] In some implementations, the specific area refers to area 3, area 4, area 6 and / or the area surrounding area 6. Any one or any combination of regions within the scope, such as region 3, region 4, region 6 and / or the area surrounding region 6. Range, Area 3 + Area 4, Area 3 + Area 6 and / or the area surrounding Area 6 Range, area 4 + area 6 and / or the area surrounding area 6 The scope, area 3 + area 4 + area 6 and / or the area surrounding area 6 Scope. In some embodiments, the method of the present invention or the α-amylase mutant does not include the area surrounding region 3, region 4, region 6 and / or region 6. Range, Area 3 + Area 4, Area 3 + Area 6 and / or the area surrounding Area 6 Range, or area 4 + area 6 and / or the area surrounding area 6 The range of amino acid changes. "+" indicates that the two are "and".

[0084] Region 3 is amino acids 117-137 of the reference *Bacillus stearothermophilus* α-amylase (SEQ ID NO:1), or, in the case of other α-amylases, the region that overlaps structurally with amino acids 117-137 of SEQ ID NO:1. In some embodiments, region 3 may be amino acids 116-136 of *Bacillus licheniformis* wild-type α-amylase (SEQ ID NO:25). In some embodiments, region 3 may be amino acids 151-171 of *Bacillus* α-amylase (SEQ ID NO:44).

[0085] In some embodiments, amino acid alterations within region 3 include the insertion, substitution, and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 3.

[0086] In some embodiments, the amino acid changes within region 3 include substitutions of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 3.

[0087] Region 4 is amino acids 176-198 of the reference *Bacillus stearothermophilus* α-amylase (SEQ ID NO:1), or, in the case of other α-amylases, the region that overlaps structurally with amino acids 176-198 of SEQ ID NO:1. In some embodiments, region 4 may be amino acids 175-195 of *Bacillus licheniformis* wild-type α-amylase (SEQ ID NO:25). In some embodiments, region 4 may be amino acids 211-233 of *Bacillus* α-amylase (SEQ ID NO:44).

[0088] In some embodiments, amino acid changes within region 4 include the insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 4.

[0089] In some embodiments, amino acid changes within region 4 include substitutions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 4.

[0090] Region 6 is amino acids 299-323 of the reference *Bacillus stearothermophilus* α-amylase (SEQ ID NO:1), or, in the case of other α-amylases, the region that overlaps structurally with amino acids 299-323 of SEQ ID NO:1. In some embodiments, region 6 may be amino acids 296-320 of *Bacillus licheniformis* wild-type α-amylase (SEQ ID NO:25). In some embodiments, region 6 may be amino acids 334-358 of *Bacillus* α-amylase (SEQ ID NO:44).

[0091] In some embodiments, amino acid alterations within region 6 include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6.

[0092] In some embodiments, the amino acid changes within region 6 include substitutions of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids within region 6.

[0093] Area 6 and surrounding The range includes a distance of no more than 1 / 3 of at least one amino acid within the range of region 6. Amino acids.

[0094] In some implementation schemes, the area surrounding Region 6 The range can include regions whose spatial distance from any amino acid within the range of region 6 is no greater than [missing value]. The amino acid, or the spatial distance between it and any two, three, four or more, or all amino acids within region 6, is no greater than [amount missing]. The spatial distances between amino acids in the three-dimensional structure of a protein can be determined based on the protein's three-dimensional structure using techniques well-known to those skilled in the art; for example, PyMol software can be used to determine the distances between amino acids based on the protein's three-dimensional structure. In some embodiments, the distances are determined around specific regions of the protein (e.g., region 6 as described in this disclosure). The range of amino acids can be determined by selecting all amino acids in a specific region in PyMol software, and then selecting "modify," "around," and "residues within." "After three steps, the amino acids hit by these steps are the amino acids surrounding a specific region of the protein (e.g., region 6 as described in this disclosure)." Amino acids within the specified range.

[0095] In some implementation schemes, the area surrounding Region 6 The range refers to amino acids 274, 277, 284, 297, 343, 359, and 406 of the reference *Bacillus stearothermophilus* α-amylase (SEQ ID NO:1), or, in the case of other α-amylases, the region (or amino acids) that overlaps in three-dimensional structure with amino acids 274, 277, 284, 297, 343, 359, and 406 of SEQ ID NO:1. In some embodiments, the region 6 is surrounded by... The range can be amino acids 271, 274, 281, 294, 340, 356, and 406 of the wild-type α-amylase of Bacillus licheniformis (SEQ ID NO:25). In some embodiments, region 6 is surrounded by... The range can be amino acids 309, 312, 319, 332, 378, 394, and 441 of Bacillus α-amylase (SEQ ID NO:44).

[0096] In some implementation schemes, around region 6 Amino acid changes within the range include those around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range.

[0097] In some implementation schemes, around region 6 Amino acid changes within the range include those around region 6. The substitution of any 1, 2, 3, 4, 5, 6 or 7 amino acids within the range.

[0098] In some embodiments, the 3D structure of the α-amylase is compared with that of a reference *Bacillus stearothermophilus* α-amylase (such as the α-amylase shown in SEQ ID NO:1) using PyMol, DaLi, CE (combinational extension), TM-align, SSAP, FLEXPROT, FATCAT, or PDB databases, and the RMSD values ​​of the comparison are obtained. In some embodiments, the comparison can use default parameters.

[0099] In some implementations, the alignment function (e.g., the Alignment-Super function) in a PyMol plugin is used to perform a three-dimensional structural alignment of the α-amylase with a reference *Bacillus stearothermophilus* α-amylase (such as the α-amylase shown in SEQ ID NO:1), and the RMSD values ​​of the alignment are obtained. In some implementations, the alignment can use default parameters. In some implementations, the parameters used are set to cycles = 5. In some implementations, the parameters used are set to cutoff = 2.0.

[0100] In some embodiments, the method and α-amylase mutant of the present invention involve amino acid alterations selected from the group consisting of:

[0101] (a) Amino acid changes within region 3, including substitutions of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids within region 3;

[0102] (b) Amino acid changes within region 4, including substitutions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids within region 4;

[0103] (c) In and / or around region 6 Amino acid alterations within the range include substitutions of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids within region 6; and / or around region 6. The substitution of any 1, 2, 3, 4, 5, 6, or 7 amino acids within the range;

[0104] (d) Amino acid changes in region 3 and region 4, wherein the amino acid changes in region 3 include substitutions of at least 7 amino acids in region 3, and the amino acid changes in region 4 include substitutions of at least 2 amino acids in region 4.

[0105] (e) Amino acid changes in region 3 and region 6, wherein the amino acid changes in region 3 include substitutions of at least 6 amino acids in region 3, and the amino acid changes in region 6 include substitutions of at least 6 amino acids in region 6.

[0106] (f) Amino acid changes within region 4 and region 6, wherein the amino acid changes within region 4 include the substitution of at least one amino acid within region 4, and the amino acid changes within region 6 include the substitution of at least two amino acids within region 6; and

[0107] (g) Within the area of ​​Zone 3, within the area of ​​Zone 4, and around Zone 6 and / or Zone 6 Amino acid changes within the specified range, wherein amino acid changes within region 3 include substitutions of at least 6 amino acids within region 3, amino acid changes within region 4 include substitutions of at least 2 amino acids within region 4, amino acid changes within region 6 include substitutions of at least 6 amino acids within region 6, and changes surrounding region 6... Amino acid changes within the range include those around region 6. The substitution of any 1-7 amino acids within the range.

[0108] In some embodiments, the method of the present invention and the α-amylase mutant may also involve amino acid alterations selected from the group consisting of:

[0109] Amino acid alterations within region 3 and region 4, wherein amino acid alterations within region 3 include substitutions of at least 7, 8, 9, 10, 11, or 12 amino acids within region 3, and amino acid alterations within region 4 include substitutions of at least 2 amino acids within region 4; and amino acid alterations within region 3 and region 4, wherein amino acid alterations within region 3 include substitutions of at least 7 amino acids within region 3, and amino acid alterations within region 4 include substitutions of at least 2, 3, 4, or 5 amino acids within region 4.

[0110] In some embodiments, the method of the present invention and the α-amylase mutant may also involve amino acid alterations selected from the group consisting of:

[0111] Amino acid changes within regions 3 and 6, wherein amino acid changes within region 3 include substitutions of at least 6, 7, 8, 9, 10, 11, or 12 amino acids within region 3, and amino acid changes within region 6 include substitutions of at least 6 amino acids within region 6; and

[0112] Amino acid changes within region 3 and region 6, wherein amino acid changes within region 3 include substitutions of at least 6 amino acids within region 3, and amino acid changes within region 6 include substitutions of at least 6, 7, 8, 9, 10, 11, or 12 amino acids within region 6.

[0113] In some embodiments, the method of the present invention and the α-amylase mutant may also involve amino acid alterations selected from the group consisting of:

[0114] Within area 3 and around area 6 Amino acid changes within the range, wherein amino acid changes within region 3 include substitutions of at least 6, 7, 8, 9, 10, 11, or 12 amino acids within region 3, and changes around region 6. Amino acid changes within the range include those around region 6. The substitution of any 1, 2, 3, 4, 5, 6 or 7 amino acids within the range.

[0115] In some embodiments, the method of the present invention and the α-amylase mutant may also involve amino acid alterations selected from the group consisting of:

[0116] Amino acid changes within regions 4 and 6, wherein amino acid changes within region 3 include substitutions of at least 1, 2, 3, 4, 5, 6, or 7 amino acids within region 4, and amino acid changes within region 6 include substitutions of at least 2 amino acids within region 6; and

[0117] Amino acid changes within region 4 and region 6, wherein amino acid changes within region 4 include the substitution of at least one amino acid within region 4, and amino acid changes within region 6 include the substitution of at least two, three, four, five, six, seven, or eight amino acids within region 6.

[0118] In some embodiments, the method of the present invention and the α-amylase mutant may also involve amino acid alterations selected from the group consisting of:

[0119] Within area 4 and around area 6 Amino acid changes within the range, wherein amino acid changes within region 4 include substitutions of at least 1, 2, 3, 4, 5, 6, or 7 amino acids within region 4, and changes around region 6. Amino acid changes within the range include those around region 6. The substitution of any 1, 2, 3, 4, 5, 6 or 7 amino acids within the range.

[0120] In some embodiments, the method of the present invention and the α-amylase mutant may also involve amino acid alterations selected from the group consisting of:

[0121] Amino acid changes within regions 3, 4 and 6, wherein amino acid changes within region 3 include substitutions of at least 6, 7, 8, 9, 10 or 11 amino acids within region 3, amino acid changes within region 4 include substitutions of at least 2 amino acids within region 4, and amino acid changes within region 6 include substitutions of at least 6 amino acids within region 6.

[0122] Amino acid changes within regions 3, 4, and 6, wherein amino acid changes within region 3 include substitutions of at least six amino acids within region 3; amino acid changes within region 4 include substitutions of at least two, three, four, five, six, seven, or eight amino acids within region 4; and amino acid changes within region 6 include substitutions of at least six amino acids within region 6; and

[0123] Amino acid changes within regions 3, 4, and 6, wherein amino acid changes within region 3 include substitutions of at least 6 amino acids within region 3, amino acid changes within region 4 include substitutions of at least 2 amino acids within region 4, and amino acid changes within region 6 include substitutions of at least 6, 7, 8, 9, 10, or 11 amino acids within region 6.

[0124] In some embodiments, the method of the present invention and the α-amylase mutant may also involve amino acid alterations selected from the group consisting of:

[0125] Within the area of ​​Zone 3, the area of ​​Zone 4, and the surrounding area of ​​Zone 6 Amino acid changes within the specified range, wherein amino acid changes within region 3 include substitutions of at least 6, 7, 8, 9, 10, or 11 amino acids within region 3; amino acid changes within region 4 include substitutions of at least 2 amino acids within region 4; and changes around region 6... Amino acid changes within the range include those around region 6. Substitution of any 1-7 amino acids within the range; and

[0126] Within the area of ​​Zone 3, the area of ​​Zone 4, and the surrounding area of ​​Zone 6 Amino acid changes within the specified range, wherein amino acid changes within region 3 include substitutions of at least 6 amino acids within region 3, amino acid changes within region 4 include substitutions of at least 2, 3, 4, 5, 6, 7, or 8 amino acids within region 4, and changes around region 6... Amino acid changes within the range include those around region 6. The substitution of any 1-7 amino acids within the range.

[0127] In some embodiments, the method of the present invention and the α-amylase mutant may also involve amino acid alterations selected from the group consisting of:

[0128] Within area 3, within area 4, and within and around area 6. Amino acid changes within the specified range, wherein amino acid changes within region 3 include substitutions of at least 5, 6, 7, 8, 9, 10, or 11 amino acids within region 3 (e.g., substitutions of 5-10, 5-7, or 6-10 amino acids); amino acid changes within region 4 include substitutions of at least 2, 3, or 4 amino acids within region 4 (e.g., substitutions of 2-4 or 3-4 amino acids); amino acid changes within region 6 include substitutions of at least 5, 6, 7, or 8 amino acids within region 6 (e.g., substitutions of 5-8 or 6-7 amino acids); and changes surrounding region 6... Amino acid changes within the range include those around region 6. The substitution of any 1-7 amino acids within the range (e.g., substitution of 4-7, 5-7, or 4-6 amino acids);

[0129] Within area 3, within area 4, and within and around area 6. Amino acid changes within the specified range, wherein amino acid changes within region 3 include substitutions of at least 6 amino acids within region 3, amino acid changes within region 4 include substitutions of at least 2, 3, 4, 5, 6, 7, or 8 amino acids within region 4, amino acid changes within region 6 include substitutions of at least 6 amino acids within region 6, and changes surrounding region 6... Amino acid changes within the range include those around region 6. Substitution of any 1-7 amino acids within the range; and

[0130] Within area 3, within area 4, and within and around area 6. Amino acid changes within the specified range, wherein amino acid changes within region 3 include substitutions of at least 6 amino acids within region 3, amino acid changes within region 4 include substitutions of at least 2 amino acids within region 4, and amino acid changes within region 6 include substitutions of at least 6, 7, 8, 9, 10, or 11 amino acids within region 6, and changes surrounding region 6. Amino acid changes within the range include those around region 6. The substitution of any 1-7 amino acids within the range.

[0131] A. Methods for modifying amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase (S-type amylase) and their mutants.

[0132] In some embodiments, the parent enzyme (or an α-amylase with a similar three-dimensional structure to the reference *Bacillus stearothermophilus* α-amylase) is an α-amylase with a similar sequence to *Bacillus stearothermophilus* α-amylase. For example, it may comprise the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:9-24, or have 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%, at least 99%, and less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:5.

[0133] In some embodiments, mutants of α-amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase contain amino acid alterations relative to the parental α-amylase, which will be described in detail below. In some embodiments, the parental α-amylase may be the mutant α-amylase shown in SEQ ID NO:5. In some embodiments, the parental α-amylase may be a wild-type α-amylase, such as the α-amylase shown in SEQ ID NO:2. In some embodiments, the parental α-amylase may be the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:9-24. In some embodiments, the parental α-amylase may be a mature polypeptide (e.g., a mature polypeptide with the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:9-24) that does not contain a signal peptide. In some embodiments, the parental α-amylase comprises a signal peptide (e.g., a polypeptide obtained by adding a signal peptide to the N-terminus of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:9-24).

[0134] In this article, unless otherwise specified, the positions of amino acids in the described α-amylases or their mutants that have similar sequences to Bacillus stearothermophilus α-amylase are determined based on the amino acid sequence shown in SEQ ID NO:1.

[0135] In some embodiments, for the modification method of an amylase having a sequence similar to that of Bacillus stearothermophilus α-amylase or a mutant thereof, the amino acid changes within region 3 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of amino acid positions 117, 118, 119, 120, 121, 122, 124, 125, 127, 128, 129, 130, 132, 133, 134, 135, 136 and 137, said amino acid position numbers corresponding to the position numbers of SEQ ID NO:1 (BSG).

[0136] In some embodiments, for methods of modifying amylases with sequences similar to those of Bacillus stearothermophilus α-amylase or mutants thereof, the amino acid substitutions within region 3 include one or more of the substitutions described below:

[0137] The amino acid at position 117 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by W, T, S, M, L, K or E; more preferably by E.

[0138] The amino acid at position 118 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably V;

[0139] The amino acid at position 119 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y or Q.

[0140] The amino acid at position 120 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D or A;

[0141] The amino acid at position 121 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by M;

[0142] The amino acid at position 122 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably D;

[0143] The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y, T, N, or F; more preferably N.

[0144] The amino acid at position 125 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably N;

[0145] The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by R or L;

[0146] The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or E.

[0147] The amino acid at position 129 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by V, P, L, K, I or A.

[0148] The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably E or D.

[0149] The amino acid at position 133 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by W, V, S, P, K, A, or E; and

[0150] The amino acid at position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by F or E.

[0151] In some embodiments, for the modification of an amylase having a sequence similar to that of Bacillus stearothermophilus α-amylase or a mutant thereof, the amino acid changes within region 4 include substitutions of any 1, 2, 3, 4, 5, 6, 7 or 8 amino acid positions from amino acid positions 176, 177, 178, 179, 184, 188, 191 and 193, the amino acid position numbers corresponding to the position numbers of SEQ ID NO:1 (BSG).

[0152] In some embodiments, for methods of modifying amylases with sequences similar to those of Bacillus stearothermophilus α-amylase or mutants thereof, amino acid substitutions within region 4 include one or more of the following substitutions:

[0153] The amino acid at position 177 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L;

[0154] The amino acid at position 184 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by Q, K or E;

[0155] The amino acid at position 179 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by S, K, E or A.

[0156] The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably P;

[0157] The amino acid at position 191 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with N, K, or D; and

[0158] The amino acid at position 193 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably K or F.

[0159] In some embodiments, for the modification method of an amylase having a sequence similar to that of Bacillus stearothermophilus α-amylase or a mutant thereof, the amino acid changes in region 6 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid positions from amino acid positions 300, 302, 304, 305, 307, 309, 310, 311, 312, 313, 316, 317, 318, 319, 321 and 322, where the amino acid position numbers correspond to the position numbers of SEQ ID NO:1 (BSG);

[0160] In some embodiments, for methods of modifying amylases with sequences similar to those of Bacillus stearothermophilus α-amylase or mutants thereof, amino acid substitutions within region 6 include one or more of the following substitutions:

[0161] The amino acid at position 304 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably S, N, K or G.

[0162] The amino acid at position 311 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by T, L, I, F or E.

[0163] The amino acid at position 312 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, K, E or D.

[0164] The amino acid at position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably replaced by V, T, L or A;

[0165] The amino acid at position 318 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by Y, T, L, K, or E.

[0166] The amino acid at position 319 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y, R, K, H, or A; and

[0167] The amino acid at position 321 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E or D; and

[0168] The amino acid at position 322 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y, K, or H.

[0169] In some embodiments, for methods of modifying amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, or mutants thereof, around region 6 The amino acid changes within the range include substitutions of any 1, 2, 3, 4, 5, 6, or 7 amino acid positions in amino acid positions 274, 277, 284, 297, 343, 359, and 406, wherein the amino acid position numbers correspond to the position numbers in SEQ ID NO:1 (BSG).

[0170] In some embodiments, for methods of modifying amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, or mutants thereof, around region 6 The amino acid substitutions within the range are selected from one or more of the following substitutions:

[0171] The amino acid at position 274 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably T, L, K or E.

[0172] The amino acid at position 277 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L;

[0173] The amino acid at position 284 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably V or I.

[0174] The amino acid at position 297 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, K or E.

[0175] The amino acid at position 343 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by Q, K or E.

[0176] The amino acid at position 359 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with D; and

[0177] The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably K.

[0178] In some embodiments, for methods of modifying amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, or their mutants, the amino acid changes include amino acid positions 18, 50, 52, 59, 104, 108, 115, 117, 118, 119, 120, 121, 122, 124, 125, 127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 143, 164, 168, 172, 176, 177, 178, 179, 184, 188, 191, 193, 204, 206, 208, 212, 241, 254, 267, 271. Substitution, insertion, and / or deletion at one or more of the following positions: 273, 274, 276, 277, 278, 281, 282, 284, 293, 294, 296, 297, 300, 302, 304, 305, 307, 309, 310, 311, 312, 313, 316, 317, 318, 319, 321, 322, 333, 339, 341, 343, 353, 358, 359, 372, 374, 375, 376, 387, 389, 392, 393, 403, 406, 416, 426, 429, 450, 452, 479, wherein the amino acid position corresponds to the position in SEQ ID NO:1.

[0179] In some embodiments, the obtained mutant has 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%, at least 99%, and less than 100% sequence identity with the amino acid sequence or mature polypeptide of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:9-24, and the mutant has α-amylase activity.

[0180] In some embodiments, the obtained mutant is sequence-aligned with the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:9-24 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide removed).

[0181] In some embodiments, the mutant of the amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase is a mature polypeptide that does not contain a signal peptide and / or a leader sequence. In some embodiments, the mutant of the amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase contains a signal peptide and / or a leader sequence. In some embodiments, the signal peptide may be encoded by a nucleotide sequence as shown in SEQ ID NO:8.

[0182] In some embodiments, for methods of modifying amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, or mutants thereof, the amino acid changes include substitutions, insertions, and / or deletions at one or more of the following positions:

[0183] The amino acid at position 18 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0184] The amino acid at position 50 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by S, A or E.

[0185] The amino acid at position 52 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0186] The amino acid at position 59 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0187] The amino acid at position 104 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V;

[0188] The amino acid at position 108 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0189] The amino acid at position 115 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or T.

[0190] The amino acid at position 117 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by E or T or W or M or K or L or S.

[0191] The amino acid at position 118 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V;

[0192] The amino acid at position 119 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y or Q.

[0193] The amino acid at position 120 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or D;

[0194] The amino acid at position 121 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably M;

[0195] The amino acid at position 122 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably D;

[0196] The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by N or F or Y or T.

[0197] The amino acid at position 125 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0198] The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L or R.

[0199] The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or E.

[0200] The amino acid at position 129 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by I or V or K or A or L or P.

[0201] The amino acid at position 130 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V or T.

[0202] The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or D.

[0203] The amino acid at position 133 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or V or W or P or A or K or S.

[0204] The amino acid at position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or F.

[0205] The amino acid at position 135 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably by E, K, D, T, N, P, W, G, M, or R.

[0206] The amino acid at position 136 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V or A.

[0207] The amino acid at position 137 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A, D or K.

[0208] The amino acid at position 138 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0209] The amino acid at position 143 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E;

[0210] The amino acid at position 164 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F or T.

[0211] The amino acid at position 168 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0212] The amino acid at position 172 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E;

[0213] The amino acid at position 176 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by F or L;

[0214] The amino acid at position 177 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0215] The amino acid at position 178 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by I;

[0216] The amino acid at position 179 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by S or E or A or K.

[0217] The amino acid at position 184 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by E, Q or K.

[0218] The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably P;

[0219] The amino acid at position 191 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D, N or K;

[0220] The amino acid at position 193 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K;

[0221] The amino acid at position 204 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V or I.

[0222] The amino acid at position 206 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0223] The amino acid at position 208 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0224] The amino acid at position 212 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably T;

[0225] The amino acid at position 241 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0226] The amino acid at position 254 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S;

[0227] The amino acid at position 267 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably T;

[0228] The amino acid at position 271 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K;

[0229] The amino acid at position 273 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably Q;

[0230] The amino acid at position 274 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by L or K or E or T.

[0231] The amino acid at position 276 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F;

[0232] The amino acid at position 277 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0233] The amino acid at position 278 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or E.

[0234] The amino acid at position 281 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D;

[0235] The amino acid at position 282 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably by Y, N, or D.

[0236] The amino acid at position 284 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V or I.

[0237] The amino acid at position 293 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0238] The amino acid at position 294 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N or R.

[0239] The amino acid at position 296 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0240] The amino acid at position 297 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by N, E or K.

[0241] The amino acid at position 300 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0242] The amino acid at position 302 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0243] The amino acid at position 304 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by K or N or G or S.

[0244] The amino acid at position 305 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0245] The amino acid at position 307 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0246] The amino acid at position 309 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by K or D or N or E.

[0247] The amino acid at position 310 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by I;

[0248] The amino acid position 311 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by L or E or I or F or T.

[0249] The amino acid at position 312 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E or K or N.

[0250] The amino acid at position 313 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or G;

[0251] The amino acid position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably replaced by V or T or L or A;

[0252] The amino acid at position 317 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R, E or S.

[0253] The amino acid at position 318 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably by Y, E, K, T, or L.

[0254] The amino acid position 319 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R or H or Y or K or A.

[0255] The amino acid at position 321 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E;

[0256] The amino acid at position 322 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by K, H or Y;

[0257] The amino acid at position 333 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably Q;

[0258] The amino acid at position 339 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E;

[0259] The amino acid at position 341 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R or Q;

[0260] The amino acid at position 343 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by E, K or Q;

[0261] The amino acid at position 353 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L or Y.

[0262] The amino acid at position 358 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S or N.

[0263] The amino acid at position 359 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably D;

[0264] The amino acid at position 372 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V;

[0265] The amino acid at position 374 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or K;

[0266] The amino acid at position 375 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or F.

[0267] The amino acid at position 376 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably G;

[0268] The amino acid at position 387 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by I;

[0269] The amino acid at position 389 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably T or K;

[0270] The amino acid at position 392 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K;

[0271] The amino acid at position 393 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N or Y.

[0272] The amino acid at position 403 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F;

[0273] The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K;

[0274] At amino acid position 416, A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V are used, preferably S is used instead;

[0275] The amino acid at position 426 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably T or V.

[0276] The amino acid at position 429 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S;

[0277] The amino acid at position 450 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E;

[0278] The amino acid at position 452 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by I;

[0279] The amino acid at position 479 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by I;

[0280] The amino acid position thereon corresponds to the position in SEQ ID NO:1.

[0281] In some embodiments, for methods of modifying amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, or mutants thereof, the resulting mutants contain one or more amino acids selected from the group consisting of: 18A, 50A, 50E, 50S, 52Y, 59A, 104V, 108A, 115K, 115T, 117E, 117K, 117L, 117M, 117S, 117T, 117W, 118V, 119Q, 119Y, 120A, 120D, 121M, 122D, 124F, 124N, 124T, 124Y, 125N, 127L, 127R, 128E, 128K, 129A, 129I, 129K, 12 9L, 129P, 129V, 130T, 130V, 132D, 132E, 133A, 133E, 133K, 133P, 133S, 133V , 133W, 134E, 134F, 135D, 135E, 135G, 135K, 135M, 135N, 135P, 135R, 135T, 1 35W, 136A, 136V, 137A, 137D, 137K, 138L, 143E, 164F, 164T, 168A, 172E, 176 F, 176L, 177L, 178I, 179A, 179E, 179K, 179S, 184E, 184K, 184Q, 188P, 191D, 1 91K, 191N, 193K, 204I, 204V, 206Y, 208N, 212T, 241Y, 254S, 267T, 271K, 273 Q, 274E, 274K, 274L, 274T, 276F, 277L, 278E, 278K, 281D, 282D, 282N, 282Y, 284I, 284V, 293Y, 294N, 294R, 296A, 297E, 297K, 297N, 300N, 302N, 304G, 30 4K, 304N, 304S, 305Y, 307L, 309D, 309E, 309K, 309N, 310I, 311E, 311F, 311I, 311L, 311T, 312D, 312E, 312K, 312N, 313D, 313G, 316A, 316L, 316T, 316V, 31 7E, 317R, 317S, 318E, 318K, 318L, 318T, 318Y, 319A, 319H, 319K, 319R, 319Y, 321D, 321E, 322H, 322K, 322Y, 333Q, 339E, 341Q, 341R, 343E, 343K, 343Q, 35 3L, 353Y, 358N, 358S, 359D, 372V, 374A, 374K, 375F, 375K, 376G, 387I, 389K,389T, 392K, 393N, 393Y, 403F, 406K, 416S, 426T, 426V, 429S, 450E, 452I, 479I, wherein the amino acid positions correspond to the positions in SEQ ID NO:1.

[0282] In some embodiments, for methods of modifying amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, or mutants thereof, the amino acid alterations include one or more substitutions selected from the group consisting of: 18A, 50A, 50E, 50S, 52Y, 59A, 104V, 108A, 115K, 115T, 117E, 117K, 117L, 117M, 117S, 117T, 117W, 118V, 119Q, 119Y, 120A, 120D, 121M, 122D, 124F, 124N, 124T, 124Y, 125N, 127L, 127R, 128E, 128K, 129A, 129I, 129K, 129L. 129P, 129V, 130T, 130V, 132D, 132E, 133A, 133E, 133K, 133P, 133S, 133V, 13 3W, 134E, 134F, 135D, 135E, 135G, 135K, 135M, 135N, 135P, 135R, 135T, 135W, 136A, 136V, 137A, 137D, 137K, 138L, 143E, 164F, 164T, 168A, 172E, 176F, 17 6L, 177L, 178I, 179A, 179E, 179K, 179S, 184E, 184K, 184Q, 188P, 191D, 191K, 191N, 193K, 204I, 204V, 206Y, 208N, 212T, 241Y, 254S, 267T, 271K, 273Q, 27 4E, 274K, 274L, 274T, 276F, 277L, 278E, 278K, 281D, 282D, 282N, 282Y, 284I, 284V, 293Y, 294N, 294R, 296A, 297E, 297K, 297N, 300N, 302N, 304G, 304K, 30 4N, 304S, 305Y, 307L, 309D, 309E, 309K, 309N, 310I, 311E, 311F, 311I, 311L, 311T, 312D, 312E, 312K, 312N, 313D, 313G, 316A, 316L, 316T, 316V, 317E, 31 7R, 317S, 318E, 318K, 318L, 318T, 318Y, 319A, 319H, 319K, 319R, 319Y, 321D, 321E, 322H, 322K, 322Y, 333Q, 339E, 341Q, 341R, 343E, 343K, 343Q, 353L, 35 3Y, 358N, 358S, 359D, 372V, 374A, 374K, 375F, 375K, 376G, 387I, 389K, 389T,392K, 393N, 393Y, 403F, 406K, 416S, 426T, 426V, 429S, 450E, 452I, 479I, wherein the amino acid positions correspond to the positions in SEQ ID NO:1.

[0283] In some embodiments, for methods of modifying amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, or mutants thereof, the amino acid alterations include one or more substitutions selected from the group consisting of: D18A, T50A, T50E, T50S, R52Y, V59A, F104V, G108A, W115K, W115T, D117E, D117K, D117L, D117M, D117S, D117T, D117W, A118V, V119Q, V119Y, E120A, E120D, V121M, N122D, S124F, S124N, S124T, S124Y, D125N, N127L, N127R. , Q128E, Q128K, E129A, E129I, E129K, E129L, E129P, E129V, I130T, I130V, G 132D, G132E, T133A, T133E, T133K, T133P, T133S, T133V, T133W, Y134E, Y134 F, Q135D, Q135E, Q135G, Q135K, Q135M, Q135N, Q135P, Q135R, Q135T, Q135W, I136A, I136V, Q137A, Q137D, Q137K, A138L, D143E, V164F, V164T, E168A, L17 2E, Y176F, Y176L, K177L, F178I, R179A, R179E, R179K, R179S, A184E, A184K , A184Q, E188P, T191D, T191K, T191N, F193K, L204I, L204V, M206Y, H208N, V2 12T, F241Y, Q254S, S267T, N271K, L273Q, H274E, H274K, H274L, H274T, Y276 F, I277L, T278E, T278K, N281D, G282D, G282N, G282Y, M284I, M284V, N293Y, K 294N, K294R, Y296A, T297E, T297K, T297N, K300N, G302N, A304G, A304K, A30 4N, A304S, F305Y, M307L, T309D, T309E, T309K, T309N, L310I, M311E, M311F, M311I, M311L, M311T, T312D, T312E, T312K, T312N, N313D, N313G, M316A, M31 6L, M316T, M316V, K317E, K317R, K317S, D318E, D318K, D318L, D318T, D318Y,Q319A, Q319H, Q319K, Q319R, Q319Y, T321D, T321E, L322H, L322K, L322Y, E333Q, Q3 39E, W341Q, W341R, D343E, D343K, D343Q, F353L, F353Y, Q358N, Q358S, E359D, I372 V, Q374A, Q374K, Y375F, Y375K, N376G, L387I, I389K, I389T, R392K, D393N, D393Y, L403F, S406K, G416S, A426T, A426V, T429S, Y450E, L452I, V479I, wherein the amino acid positions correspond to the positions in SEQ ID NO:1. In some embodiments, for methods of modifying amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, or mutants thereof, the resulting mutants contain the above substitutions relative to the amino acid sequence shown in SEQ ID NO:5.

[0284] In some embodiments, for methods of modifying amylases with a sequence similar to that of Bacillus stearothermophilus α-amylase or mutants thereof, the amino acid alterations may include substitutions at one or more of the following positions: amino acid positions 59, 184, 188, 193, 254, 284, 293, 297 and / or 416. In some embodiments, for methods of modifying amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, or mutants thereof, the amino acid alterations may include one or more substitutions selected from the group consisting of: a substitution at amino acid position 59 of 59A; a substitution at amino acid position 184 of 184E, 184Q, or 184K; a substitution at amino acid position 188 of 188P; a substitution at amino acid position 193 of 193K; a substitution at amino acid position 254 of 254S; a substitution at amino acid position 284 of 284V or 284I; a substitution at amino acid position 293 of 293Y; and / or a substitution at amino acid position 416 of 416S. In some embodiments, for the modification method of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase, or a mutant thereof, the resulting mutant may contain one or more amino acids selected from the group consisting of: amino acid 59A at amino acid position 59; amino acid 184E, 184Q, or 184K at amino acid position 184; amino acid 188P at amino acid position 188; amino acid 193K at amino acid position 193; amino acid 254S at amino acid position 254; amino acid 284V or 284I at amino acid position 284; amino acid 293Y at amino acid position 293; and / or amino acid 416S at amino acid position 416. In some embodiments, for the modification of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the amino acid alteration may include one or more substitutions selected from the group consisting of: a substitution at amino acid position 59 of V59A; a substitution at amino acid position 184 of A184E, A184Q, or A184K; a substitution at amino acid position 188 of E188P; a substitution at amino acid position 193 of N193K; a substitution at amino acid position 254 of Q254S; a substitution at amino acid position 284 of M284V or M284I; a substitution at amino acid position 293 of N293Y; and / or a substitution at amino acid position 416 of V416S.

[0285] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, the amino acid alteration may further include substitutions at one or more positions of amino acid positions 122, 124, 128, 129, 133, 135, 191, 212, and / or 281. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, the amino acid alteration may further include substitutions selected from the group consisting of: a substitution at amino acid position 122 of 122D; a substitution at amino acid position 124 of 124N, 124F, 124Y, or 124T; a substitution at amino acid position 128 of 128K or 128E; and a substitution at amino acid position 129 of 129I, 129V, or 129E. 129K, 129A, 129L, or 129P; substitutions at amino acid position 133 are: 133E, 133V, 133W, 133P, 133A, 133K, or 133S; substitutions at amino acid position 135 are: 135E, 135K, 135D, 135T, 135N, 135P, 135W, 135G, 135M, or 135R; substitutions at amino acid position 191 are: 191D, 191N, or 191K; substitutions at amino acid position 212 are: 212T; and / or substitutions at amino acid position 281 are: 281D. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the resulting mutant may further comprise one or more amino acids selected from the group consisting of: amino acid 122D at amino acid position 122; amino acid 124N, 124F, 124Y, or 124T at amino acid position 124; amino acid 128K or 128E at amino acid position 128; and amino acid 129I, 129V, ... 129K, 129A, 129L, or 129P; amino acids at position 133 are: 133E, 133V, 133W, 133P, 133A, 133K, or 133S; amino acids at position 135 are: 135E, 135K, 135D, 135T, 135N, 135P, 135W, 135G, 135M, or 135R; amino acids at position 191 are: 191D, 191N, or 191K; amino acids at position 212 are: 212T; and / or amino acids at position 281 are: 281D.

[0286] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, the amino acid alteration may further include a group of substitutions or a group of amino acids selected from any of the following:

[0287] 59A / 188P / 254S / 293Y;

[0288] 59A / 188P / 254S / 293Y / 212T;

[0289] 59A / 188P / 254S / 293Y / 281D; and

[0290] 59A / 188P / 254S / 293Y / 212T / 281D.

[0291] In some embodiments, based on any one of the amino acids or amino acid substitutions or combinations thereof described above, for methods of modifying amylases with a sequence similar to that of Bacillus stearothermophilus α-amylase or mutants thereof, the amino acid alteration may further include a group of substitutions selected from any one of the following:

[0292] V59A / E188P / Q254S / N293Y;

[0293] V59A / E188P / Q254S / N293Y / V212T;

[0294] V59A / E188P / Q254S / N293Y / N281D; and

[0295] V59A / E188P / Q254S / N293Y / V212T / N281D.

[0296] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration of a method for modifying an amylase with a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof may further include a substitution at amino acid position 179. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration of a method for modifying an amylase with a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof may further include the following substitutions: 179S, 179E, 179A, or 179K. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration of a method for modifying an amylase with a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof may further include the following amino acids: 179S, 179E, 179A, or 179K.

[0297] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the amino acid alteration may further include substitutions at one or more positions of amino acid positions 129, 191, 212, and / or 281. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the amino acid alteration may further include substitutions selected from the group consisting of: substitution at amino acid position 129 as 129V or 129I; substitution at amino acid position 191 as 191N or 191D; substitution at amino acid position 212 as 212T; and / or substitution at amino acid position 281 as 281D. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a similar sequence to Bacillus stearothermophilus α-amylase or a mutant thereof, the resulting mutant may further include one or more amino acids selected from the group consisting of: amino acid at amino acid position 129 being 129V or 129I; amino acid at amino acid position 191 being 191N or 191D; amino acid at amino acid position 212 being 212T; and / or amino acid at amino acid position 281 being 281D.

[0298] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with a sequence similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the amino acid alteration may further include substitutions at one or more positions of amino acid positions 304, 311, and / or 319. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with a sequence similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the amino acid alteration may further include substitutions selected from the group consisting of: substitution at amino acid position 304 of 304K, 304N, 304G, or 304S; substitution at amino acid position 311 of 311L; and / or substitution at amino acid position 319 of 319H, 319Y, or 319K. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a similar sequence to Bacillus stearothermophilus α-amylase or a mutant thereof, the resulting mutant may further include one or more amino acids selected from the group consisting of: amino acid at amino acid position 304 being 304K, 304N, 304G or 304S; amino acid at amino acid position 311 being 311L; and / or amino acid at amino acid position 319 being 319H, 319Y or 319K.

[0299] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of amylases with a sequence similar to that of *Bacillus stearothermophilus* α-amylase or its mutants, the amino acid changes may further include amino acid positions 18, 50, 52, 104, 108, 115, 117, 119, 120, 122, 124, 128, 130, 132, 133, 134, 135, 137, 143, 168, 176, 204, 206, 208. Substitution at one or more of the following positions: 267, 271, 273, 274, 276, 277, 278, 282, 296, 300, 302, 305, 307, 309, 310, 312, 313, 316, 317, 318, 321, 322, 333, 339, 341, 343, 358, 359, 372, 374, 375, 376, 387, 389, 392, 393, 403, 406, 426, 429, 450, 452, 479. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of amylases with a sequence similar to that of *Bacillus stearothermophilus* α-amylase or its mutants, the amino acid alteration may further include one or more substitutions selected from the group consisting of: substitution at amino acid position 18: 18A; substitution at amino acid position 50: 50S, 50A, or 50E; substitution at amino acid position 52: 52Y; substitution at amino acid position 104: 104V; substitution at amino acid position 108: 108A; substitution at amino acid position 115: 115K or 115T; substitution at amino acid position 117: 117K, 117E, 117L, or 117M; substitution at amino acid position 119: 119Y; substitution at amino acid position 120: 120A; substitution at amino acid position 1... The substitution at amino acid position 22 is 122D; the substitution at amino acid position 124 is 124N; the substitution at amino acid position 128 is 128K; the substitution at amino acid position 130 is 130T or 130V; the substitution at amino acid position 132 is 132D or 132E; the substitution at amino acid position 133 is 133V, 133E, 133A, 133K, or 133S; and the substitution at amino acid position 134 is 1... 34E or 134F; substitution at amino acid position 135: 135E, 135K, 135P or 135D; substitution at amino acid position 137: 137A, 137D, 137K; substitution at amino acid position 143: 143E; substitution at amino acid position 168: 168A; substitution at amino acid position 176: 176F; substitution at amino acid position 204: 204V or 204I;The substitution at amino acid position 206 is 206Y; the substitution at amino acid position 208 is 208N; the substitution at amino acid position 267 is 267T; the substitution at amino acid position 271 is 271K; the substitution at amino acid position 273 is 273Q; the substitution at amino acid position 274 is 274E, 274K, or 274L; the substitution at amino acid position 276 is 276F; the substitution at amino acid position 277 is 277L; the substitution at amino acid position 278 is 278K or 278E; the substitution at amino acid position 282 is 282Y; the substitution at amino acid position 296 is 296A; and the substitution at amino acid position 300 is... The substitutions at amino acid position 302 are: 302N; at amino acid position 305 are: 305Y; at amino acid position 307 are: 307L; at amino acid position 309 are: 309D or 309K; at amino acid position 310 are: 310I; at amino acid position 312 are: 312D, 312K, 312N, or 312E; at amino acid position 313 are: 313D or 313G; at amino acid position 316 are: 316T, 316V, or 316A; at amino acid position 317 are: 317R, 317S, or 317E; at amino acid position 318 are: 300N; at amino acid position 319 are: 300N; at amino acid position 300N are: 300N; at amino acid position 300N are: 300N; at amino acid position 300N are: 300N; at amino acid position 300N are: 300N; at amino acid position 300N are: 300N; at amino acid position 300N are: 300N; at amino acid position 319 ... The substitutions are: 318Y, 318E, 318T, or 318K; the substitution at amino acid position 321 is: 321D; the substitution at amino acid position 322 is: 322K; the substitution at amino acid position 333 is: 333Q; the substitution at amino acid position 339 is: 339E; the substitution at amino acid position 341 is: 341R or 341Q; the substitution at amino acid position 343 is: 343K or 343E; the substitution at amino acid position 358 is: 358S or 358N; the substitution at amino acid position 359 is: 359D; the substitution at amino acid position 372 is: 372V; and the substitution at amino acid position 374 is: 374A or 374K. The substitutions at amino acid position 375 are: 375K or 375F; at amino acid position 376 are: 376G; at amino acid position 387 are: 387I; at amino acid position 389 are: 389T or 389K; at amino acid position 392 are: 392K, 393N, or 393Y; at amino acid position 403 are: 403F; at amino acid position 406 are: 406K; at amino acid position 426 are: 426T or 426V; at amino acid position 429 are: 429S; at amino acid position 450 are: 450E; at amino acid position 452 are: 452I.And / or the substitution at amino acid position 479 is: 479I. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the resulting mutant may further comprise one or more amino acids selected from the group consisting of: amino acid at position 18: 18A; amino acid at position 50: 50S, 50A, or 50E; amino acid at position 52: 52Y; amino acid at position 104: 104V; amino acid at position 108: 108A; amino acid at position 115: 115K or 115T; amino acid at position 117... The amino acids at position 117 are: 117K, 117E, 117L, or 117M; the amino acid at position 119 is: 119Y; the amino acid at position 120 is: 120A; the amino acid at position 122 is: 122D; the amino acid at position 124 is: 124N; the amino acid at position 128 is: 128K; the amino acid at position 130 is: 130T or 130V; the amino acid at position 132 is: 132D or 132E; the amino acid at position 133 is: 133V, 133E, 133A, 133K, or 133S; the amino acid at position 134 is: 134E or 134F; The amino acids at position 135 are: 135E, 135K, 135P, or 135D; at position 137 are: 137A, 137D, or 137K; at position 143 is: 143E; at position 168 is: 168A; at position 176 is: 176F; at position 204 is: 204V or 204I; at position 206 is: 206Y; at position 208 is: 208N; at position 267 is: 267T; at position 271 is: 271K; and at position 273 is: 2... 73Q; Amino acids at position 274 are: 274E, 274K, or 274L; Amino acids at position 276 are: 276F; Amino acids at position 277 are: 277L; Amino acids at position 278 are: 278K or 278E; Amino acids at position 282 are: 282Y; Amino acids at position 296 are: 296A; Amino acids at position 300 are: 300N; Amino acids at position 302 are: 302N; Amino acids at position 305 are: 305Y; Amino acids at position 307 are: 307L; Amino acids at position 309 are: 309D or 309K;The amino acid at position 310 is 310I; the amino acid at position 312 is 312D, 312K, 312N, or 312E; the amino acid at position 313 is 313D or 313G; the amino acid at position 316 is 316T, 316V, or 316A; the amino acid at position 317 is 317R, 317S, or 317E; and the amino acid at position 318 is 318Y or 318E. 318T or 318K; the amino acid at position 321 is 321D; the amino acid at position 322 is 322K; the amino acid at position 333 is 333Q; the amino acid at position 339 is 339E; the amino acid at position 341 is 341R or 341Q; the amino acid at position 343 is 343K or 343E; the amino acid at position 358 is 358S or 358N; The amino acid at position 359 is 359D; the amino acid at position 372 is 372V; the amino acid at position 374 is 374A or 374K; the amino acid at position 375 is 375K or 375F; the amino acid at position 376 is 376G; the amino acid at position 387 is 387I; the amino acid at position 389 is 389T or 389K; the amino acid at position 392... The amino acids are: 392K, 393N, or 393Y; the amino acid at position 403 is: 403F; the amino acid at position 406 is: 406K; the amino acid at position 426 is: 426T or 426V; the amino acid at position 429 is: 429S; the amino acid at position 450 is: 450E; the amino acid at position 452 is: 452I; and / or the amino acid at position 479 is: 479I.

[0300] In some embodiments, based on any of the amino acid or amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, the amino acid alteration may further include a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0301] V59A / A184Q / E188P / Q254S / N293Y;

[0302] V59A / A184Q / E188P / Q254S / N293Y / N281D / V212T;

[0303] V59A / A184Q / E188P / Q254S / N293Y / N281D / V212T / E129V / T297N / R179S / T191N / M284V.

[0304] In some embodiments, based on any of the amino acid or amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, the amino acid alteration may further include a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0305] V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E18 8P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / I372V / Q374A / Y375K;

[0306] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N29 3Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y / I372V / Q374A / Y375K;

[0307] V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q25 4S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y / I372V / Q374A / Y375K;

[0308] V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304G / M311L / T312N / N313G / M316V / K317S / Q319H / I372V / Q374A / Y375K;

[0309] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y / I372V / Q374A / Y375K;

[0310] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304G / M311L / T312N / N313G / M316V / K317S / Q319H / I372V / Q374A / Y375K;

[0311] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304G / M311L / T312N / N313G / M316V / K317S / Q319H / I372V / Q374A / Y375K;

[0312] V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y;

[0313] V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304G / M311L / T312N / N313G / M316V / K317S / Q319H;

[0314] V59A / D117M / N122D / S124N / Q128K / E129V / T133E / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / T312D / M316T / K317R / Q319Y / I372V / Q374A / Y375K;

[0315] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304S / F305Y / M311L / T312D / M316T / Q319Y / I372V / Q374A / Y375K;

[0316] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304S / F305Y / M311L / T312D / M316T / D318E / Q319Y;

[0317] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / T312D / M316T / D318Y / Q319Y;

[0318] V59A / D117M / N122D / S124N / Q128K / E129V / T133E / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / T312E / M316T / K317R / D318E / Q319H;

[0319] V59A / W115T / V119Y / N122D / S124N / Q128K / E129I / G132D / T133V / Q135P / Q137D / E168A / R179K / A184Q / E188P / T191N / F193K / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0320] V59A / W115K / V119Y / N122D / S124N / Q128K / E129I / G132E / T133V / Q135P / Q137D / E168A / R179K / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0321] V59A / W115K / D117L / V119Y / N122D / S124N / Q128K / E129I / I130V / G132D / T133V / Q135E / Q137D / E168A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0322] V59A / W115K / D117L / V119Y / N122D / S124N / Q128K / E129I / I130T / G132E / T133V / Q135E / Q137D / E168A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0323] V59A / W115K / D117L / V119Y / N122D / S124N / Q128K / E129I / G132D / T133V / Q135E / Q137D / E168A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0324] V59A / W115K / D117K / V119Y / E120A / N122D / S124N / Q128K / E129I / G132D / T133V / Y134E / Q135D / Q137D / E168A / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0325] V59A / N122D / S124N / Q128K / G132D / T133V / Q135K / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0326] V59A / D117K / N122D / S124N / Q128K / E129V / G132D / T133E / Q135E / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0327] V59A / N122D / S124N / Q128K / G132E / T133V / Q135K / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0328] V59A / D117K / N122D / S124N / Q128K / G132E / T133V / Q135E / Q137A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0329] V59A / D117K / N122D / S124N / Q128K / E129I / G132E / T133V / Q135E / Q137A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0330] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274K / I277L / N281D / G282Y / M284I / N293Y / T297E / K300N / A304K / F305Y / T309D / M311L / T312K / N313D / K317E / D318E / Q319Y / T321D / L322K / D343K / E359D / S406K;

[0331] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / Y296A / T297E / A304K / M311L / T312D / M316T / D318Y / Q319H / T321D / L322K / D343E / E359D / S406K;

[0332] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / T297E / A304K / F305Y / M311L / T312D / M316T / D318Y / Q319H / T321D / D343E / E359D / S406K;

[0333] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / Y296A / T297E / K300N / A304K / M311L / T312D / M316T / D318Y / Q319H / T321D / D343K / E359D / L403F / S406K;

[0334] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / T297E / K300N / A304K / M311L / T312D / D318Y / Q319H / T321D / D343K / E359D / L403F / S406K;

[0335] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / T297E / A304K / F305Y / M311L / T312D / M316A / D318Y / Q319Y / T321D / L322K / S406K;

[0336] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319H / T321D / S406K;

[0337] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / Y296A / T297E / A304K / F305Y / M311L / T312D / M316V / D318Y / Q319H / T321D / L322K / S406K;

[0338] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / N313D / M316T / K317R / D318Y / Q319Y / T321D / L322K / S406K;

[0339] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / Y296A / T297E / A304K / T309K / M311L / T312D / N313D / M316T / D318Y / Q319Y / T321D / L322K / S406K;

[0340] V59A / E129V / R179S / A184Q / E188P / V212T / Q254S / S267T / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318T / Q319H / L322K;

[0341] V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / S267T / N271K / H274E / I277L / T278E / M284I / N293Y / T297N / N313D / D318Y / Q319H / L322K;

[0342] V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / S267T / H274L / I277L / T278K / M284I / N293Y / T297N / N313D / D318Y / Q319H;

[0343] V59A / E129V / R179S / A184Q / E188P / V212T / Q254S / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318T / Q319H / L322K;

[0344] V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / H274K / I277L / T278E / M284I / N293Y / T297N / N313D / D318Y / Q319Y / L322K;

[0345] V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318Y / Q319H;

[0346] V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / K317R / Q319Y / I372V / Q374A / Y375K;

[0347] V59A / D117E / N122D / S124N / Q128K / E129V / T133A / Q135K / Q137A / R179S / A184Q / E188P / T191N / V21 2T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / D318E / Q319H / I372V / Q374A / Y375K;

[0348] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / Q319Y / I372V / Q374A / Y375K;

[0349] V59A / D117E / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137K / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / T312K / N313D / M316V / K317R / Q319Y / I372V / Q374A / Y375K;

[0350] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304S / M311L / T312D / M316T / K317R / Q319Y / I372V / Q374A / Y375K / N376G;

[0351] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137K / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y / I372V / Q374A / Y375K;

[0352] V59A / D117E / N122D / S124N / Q128K / E129V / T133K / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / D318E / Q319H;

[0353] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / D318E / Q319H;

[0354] V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / T312K / N313D / M316T / K317R / D318E / Q319H;

[0355] V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312E / N313D / M316T / K317R / Q319Y;

[0356] V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Y134F / Q135E / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / T312D / K317R / Q319Y;

[0357] V59A / D117E / N122D / S124N / Q128K / E129V / T133S / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / T312D / D318E / Q319H;

[0358] T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204V / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N;

[0359] T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204I / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N;

[0360] T50A / V59A / F104V / G108A / E129V / D143E / R179S / A184Q / E188P / T191D / L204V / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N;

[0361] T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204I / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N;

[0362] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304N / F305Y / M311L / Q358S / E359D / I389T / D393N / S406K / G416S / A426T / T429S / Y450E / L452I;

[0363] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304N / F305Y / M311L / Q358S / E359D / I389T / D393N / S406K / G416S / A426V / Y450E / L452I / V479I;

[0364] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / Q358S / E359D / L387I / I389T / D393N / S406K / G416S / A426V / T429S / Y450E / L452I / V479I;

[0365] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304N / F305Y / M311L / Q358S / E359D / I389T / R392K / D393N / S406K / G416S / A426T / Y450E / L452I;

[0366] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304N / F305Y / M311L / Q358S / E359D / I389K / S406K / G416S / A426V / Y450E / L452I;

[0367] T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204V / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N / A304N / F305Y / M311L / Q358N / E359D / I389K / R392K / D393Y / S406K / G416S / A426T / Y450E / L452I;

[0368] T50A / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / L204V / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N / A304N / F305Y / M311L / Q358S / E359D / I389T / D393N / S406K / G416S / A426T / T429S / Y450E / L452I;

[0369] T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204I / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N / A304K / F305Y / M311L / Q358N / E359D / I389K / S406K / G416S / A426V / T429S / Y450E / L452I;

[0370] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N271K / H274E / T27 8E / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / D318Y / Q319H;

[0371] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N271K / H274E / T278K / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / N313D / M316T / D318Y / Q319H;

[0372] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274L / T278E / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / D318Y / Q319K;

[0373] V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / S267T / N271K / L273Q / H274E / Y276F / T278K / N281D / M284V / N293Y / T297N / G302N / A304K / M307L / L310I / M311L / T312K / N313D / K317E / D318K / Q319H;

[0374] D18A / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341R / I372V / Q374K / Y375F;

[0375] D18A / T50E / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341R / I372V / Q374K; or

[0376] D18A / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341Q / I372V / Q374A / Y375K.

[0377] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or their mutants, the amino acid alteration may further include substitutions at one or more positions of amino acid positions 177, 212, and / or 281. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or their mutants, the amino acid alteration may further include substitutions selected from the group consisting of 177L, 212T, and 281D. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or their mutants, the resulting mutant may further include one or more amino acids selected from the group consisting of 177L, 212T, and 281D. In some embodiments, for methods of modifying amylases with sequences similar to those of Bacillus stearothermophilus α-amylase or mutants thereof, the amino acid alterations may include one or more substitutions selected from the group consisting of: D177L, V212T, N281D.

[0378] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of an amylase having a sequence similar to that of Bacillus stearothermophilus α-amylase or a mutant thereof, the amino acid alteration may further include substitutions at one or more of the following positions: 117, 122, 124, 128, 129, 133, 135, 176 and / or 191. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of amylases with a sequence similar to that of *Bacillus stearothermophilus* α-amylase or its mutants, the amino acid alteration may include one or more substitutions selected from the group consisting of: substitution at amino acid position 117 of 117E, 117T, 117W, 117M, 117S, 117L, or 117K; substitution at amino acid position 122 of 122D; substitution at amino acid position 124 of 124N, 124F, 124Y, or 124T; and substitution at amino acid position 128 of 124N, 124F, 124Y, or 124T. The substitutions at amino acid position 129 are: 128K or 128E; the substitutions at amino acid position 129 are: 129I, 129K, 129V, 129A, 129L or 129P; the substitutions at amino acid position 133 are: 133E, 133W, 133V, 133P or 133A; the substitutions at amino acid position 135 are: 135E, 135K, 135T, 135D, 135N, 135P, 135W, 135G, 135M or 135R; the substitutions at amino acid position 176 are: 176F or 176L; and / or the substitutions at amino acid position 191 are: 191D, 191N or 191K.In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the resulting mutant may further comprise one or more amino acids selected from the group consisting of: amino acid at amino acid position 117 being: 117E, 117T, 117W, 117M, 117S, 117L, or 117K; amino acid at amino acid position 122 being: 122D; amino acid at amino acid position 124 being: 124N, 124F, 124Y, or 124T; amino acid at amino acid position 128 being: 124N, 124F, 124Y, or 124T; amino acid at amino acid position 128 being: 124N, 124F, 124Y, or 124T; amino acid at amino acid position 128 being: 124N, 124F, 124Y, or 124T; amino acid at amino acid position 128 being: 124N, 124F, 124Y, or 124T; amino acid at amino acid position 124 ... The amino acids at position 129 are: 128K or 128E; the amino acids at position 129 are: 129I, 129K, 129V, 129A, 129L or 129P; the amino acids at position 133 are: 133E, 133W, 133V, 133P or 133A; the amino acids at position 135 are: 135E, 135K, 135T, 135D, 135N, 135P, 135W, 135G, 135M or 135R; the amino acids at position 176 are: 176F or 176L; and / or the amino acids at position 191 are: 191D, 191N or 191K.

[0379] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration for methods of modifying or mutants of amylases with a sequence similar to that of *Bacillus stearothermophilus* α-amylase may further include a substitution at amino acid position 274. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration for methods of modifying or mutants of amylases with a sequence similar to that of *Bacillus stearothermophilus* α-amylase may further include the substitution 274L. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration for methods of modifying or mutants of amylases with a sequence similar to that of *Bacillus stearothermophilus* α-amylase may further include the amino acid 274L.

[0380] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of amylases with a sequence similar to that of Bacillus stearothermophilus α-amylase or its mutants, the amino acid alteration may further include substitutions at one or more of the following positions: 118, 119, 120, 121, 125, 127, 130, 132, 134, 136, 138, 164, 172, 178, 208, 241, 267, 277, 278, 282, 294, 300, 304, 309, 311, 312, 313, 316, 317, 318, 319, 321, 322, 343, 353, 359, 403, and / or 406. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of *Bacillus stearothermophilus* α-amylase, the amino acid alteration may further include one or more substitutions selected from the group consisting of: substitution at amino acid position 118: 118V; substitution at amino acid position 119: 119Q or 119Y; substitution at amino acid position 120: 120D or 120A; substitution at amino acid position 121... The substitutions at amino acid position 125 are: 125N; at amino acid position 127, the substitutions are: 127L or 127R; at amino acid position 130, the substitutions are: 130V or 130T; at amino acid position 132, the substitutions are: 132D or 132E; at amino acid position 134, the substitutions are: 134E or 134F; at amino acid position 136, the substitutions are: 136V or 136A; at amino acid position 138, the substitution is: 138L; at amino acid position 164, the substitution is: 164F or 1 64T; substitution at amino acid position 172: 172E; substitution at amino acid position 178: 178I; substitution at amino acid position 208: 208N; substitution at amino acid position 241: 241Y; substitution at amino acid position 267: 267T; substitution at amino acid position 274: 274L, 274K, 274E, or 274T; substitution at amino acid position 277: 277L; substitution at amino acid position 278: 278K or 278E; substitution at amino acid position 28... The substitutions for amino acid 2 are: 282Y, 282N, or 282D; the substitutions for amino acid 294 are: 294N or 294R; the substitution for amino acid 300 is: 300N; the substitution for amino acid 304 is: 304K; the substitutions for amino acid 309 are: 309K, 309N, 309D, or 309E; the substitutions for amino acid 311 are: 311L, 311E, 311I, 311F, or 311T; and the substitutions for amino acid 312 are: 312D, 312E, or 312K.The substitutions at amino acid position 313 are: 313D or 313G; the substitutions at amino acid position 316 are: 316V, 316T, or 316L; the substitutions at amino acid position 317 are: 317R or 317E; the substitutions at amino acid position 318 are: 318Y, 318E, 318K, 318L, or 318T; the substitutions at amino acid position 319 are: 319R, 319H, 319K, 319A, or 319Y; at amino acid position... The substitution at position 321 is 321D or 321E; the substitution at position 322 is 322K, 322H or 322Y; the substitution at position 343 is 343E, 343K or 343Q; the substitution at position 353 is 353L or 353Y; the substitution at position 359 is 359D; the substitution at position 403 is 403F; and / or the substitution at position 406 is 406K. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the resulting mutant may further comprise one or more amino acids selected from the group consisting of: amino acid at position 118: 118V; amino acid at position 119: 119Q or 119Y; amino acid at position 120: 120D or 120A; amino acid at position 121: 121M; amino acid at position 125: 125N; amino acid at position 127: 127L or 127R; amino acid at position 130: 130V or 130T; amino acid at position 132: 132D or 132E; amino acid at position 134: 134E or 134F; amino acid at position 136: ... Amino acid at position 136V or 136A; amino acid at position 138 is 138L; amino acid at position 164 is 164F or 164T; amino acid at position 172 is 172E; amino acid at position 178 is 178I; amino acid at position 208 is 208N; amino acid at position 241 is 241Y; amino acid at position 267 is 267T; amino acid at position 274... The amino acids at position 274 are: 274L, 274K, 274E, or 274T; the amino acid at position 277 is: 277L; the amino acid at position 278 is: 278K or 278E; the amino acid at position 282 is: 282Y, 282N, or 282D; the amino acid at position 294 is: 294N or 294R; the amino acid at position 300 is: 300N; and the amino acid at position 304 is: 304K.The amino acids at position 309 are: 309K, 309N, 309D, or 309E; the amino acids at position 311 are: 311L, 311E, 311I, 311F, or 311T; the amino acids at position 312 are: 312D, 312E, or 312K; the amino acids at position 313 are: 313D or 313G; the amino acids at position 316 are: 316V, 316T, or 316L; the amino acids at position 317 are: 317R or 317E; and the amino acids at position 318 are: 318Y, 318E, 318K, or 318L. Or 318T; amino acids at position 319 are: 319R, 319H, 319K, 319A, or 319Y; amino acids at position 321 are: 321D or 321E; amino acids at position 322 are: 322K, 322H, or 322Y; amino acids at position 343 are: 343E, 343K, or 343Q; amino acids at position 353 are: 353L or 353Y; amino acids at position 359 are: 359D; amino acids at position 403 are: 403F; and / or amino acids at position 406 are: 406K.

[0381] In some embodiments, based on any of the amino acid or amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, the amino acid alteration may further include a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0382] V59A / K177L / E188P / V212T / Q254S / N281D / N293Y;

[0383] V59A / E129V / K177L / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0384] V59A / K177L / E188P / V212T / Q254S / N281D / N293Y / Y176F / N122D / A184E / Q128K / T191D; and

[0385] V59A / K177L / E188P / V212T / Q254S / N281D / N293Y / Y176F / N122D / A184E / Q128K / T191D / S124N.

[0386] In some embodiments, based on any of the amino acid or amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, the amino acid alteration may further include a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0387] V59A / E129I / L172E / Y176F / K177L / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0388] V59A / A118V / E129I / I136A / A138L / V164F / L172E / K177L / F178I / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0389] V59A / A118V / V119Q / D125N / E129I / I136A / A138L / V164F / L172E / Y176L / K177L / F178I / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N;

[0390] V59A / V119Q / E129I / I136V / V164T / L172E / Y176F / K177L / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N;

[0391] V59A / D125N / N127L / E129I / I136V / V164F / L172E / K177L / A184Q / E188P / T191D / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N;

[0392] V59A / D125N / N127L / E129I / I136V / V164F / L172E / K177L / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N;

[0393] V59A / V119Q / E129I / I136V / V164T / L172E / Y176L / K177L / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0394] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / I130V / G132E / T133W / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0395] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0396] V59A / D117M / V119Y / N122D / S124N / D125N / N127L / Q128K / E129I / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0397] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0398] V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0399] V59A / D117E / N122D / S124N / Q128K / E129I / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274K / T278K / N281D / M284V / N293Y / T297N;

[0400] V59A / D117M / V119Y / N122D / S124N / D125N / N127L / Q128K / E129I / I130V / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / V212T / Q254S / S267T / H274K / T278E / N281D / M284V / N293Y / T297N;

[0401] V59A / D117E / V119Q / N122D / S124N / Q128K / E129I / G132E / T133W / Q135K / Y176F / K177L / A184E / E188P / T191K / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N;

[0402] V59A / D117L / N122D / S124N / Q128K / E129I / T133E / Q135W / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274K / T278K / N281D / M284V / N293Y / T297N;

[0403] V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N;

[0404] V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N;

[0405] V59A / D117M / N122D / S124N / D125N / N127L / Q128K / E129I / T133E / Q135E / Y17 6F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0406] V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / T278E / N281D / M284V / N293Y / T297N;

[0407] V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / T278K / N281D / M284V / N293Y / T297N;

[0408] V59A / D117L / N122D / S124N / Q128K / E129I / T133E / Q135W / Y176F / K177L / A18 4E / E188P / T191D / V212T / Q254S / H274K / T278K / N281D / M284V / N293Y / T297N;

[0409] V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / V212T / Q254S / H274L / I277L / T278K / N281D / M284V / N293Y / T297N;

[0410] V59A / E129V / Y176F / K177L / A184E / E188P / V212T / Q254S / H274K / I277L / T278K / N281D / M284V / N293Y / T297N / A304K / T309K / M311I / T312D / M316L / K317R / D318E / Q319H;

[0411] V59A / E129V / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / H274L / T278E / N281D / M284V / N293Y / T297N / A304K / T309N / M311I / T312K / N313D / K317E / D318K / Q319K;

[0412] V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / T278K / N281D / M284V / N293Y / T297N / A304K / M311T / T312E / K317R / D318E / Q319H;

[0413] V59A / D117T / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / T278E / N281D / M284V / N293Y / T297N / A304K / T309N / M311L / T312K / N313D / K317E / D318K / Q319H;

[0414] V59A / D117M / N122D / S124N / D125N / N127L / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / V212T / Q254S / H274K / T278E / N281D / M284V / N293Y / T297N / A304K / T309K / M311F / T312E / K317R / D318E / Q319H;

[0415] V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184K / E188P / V212T / Q254S / H274K / T278K / N281D / M284V / N293Y / T297N / A304K / M311I / T312D / K317R / D318E / Q319H;

[0416] V59A / D117E / N122D / S124N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0417] V59A / D117E / N122D / S124N / N127L / Q128K / E129L / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0418] V59A / D117E / N122D / S124N / D125N / N127L / Q128E / E129K / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0419] V59A / D117E / N122D / S124N / Q128K / E129P / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0420] V59A / D117E / N122D / S124Y / D125N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0421] V59A / D117E / N122D / S124N / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0422] V59A / D117M / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0423] V59A / D117E / N122D / S124T / D125N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0424] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0425] V59A / D117E / N122D / S124N / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0426] V59A / D117E / N122D / S124F / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0427] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0428] V59A / D117E / N122D / S124N / N127L / Q128K / E129P / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0429] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129P / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0430] V59A / D117M / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0431] V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0432] V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135T / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0433] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129I / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0434] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133V / Q135N / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0435] V59A / D117E / N122D / S124F / N127R / Q128K / E129I / T133P / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0436] V59A / D117M / N122D / S124N / D125N / Q128K / E129I / T133E / Q135P / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0437] V59A / D117T / N122D / S124N / D125N / N127L / Q128E / E129L / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0438] V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0439] V59A / D117S / N122D / S124N / Q128K / E129L / T133E / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0440] V59A / D117E / N122D / S124N / N127R / Q128K / E129V / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0441] V59A / D117E / N122D / S124N / Q128K / E129I / T133E / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0442] V59A / D117W / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0443] V59A / D117W / N122D / S124N / N127R / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0444] V59A / D117E / N122D / S124N / N127L / Q128K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0445] V59A / D117E / N122D / S124N / N127R / Q128K / E129V / T133P / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0446] V59A / D117W / N122D / S124N / Q128K / E129V / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319H / T321D / D343E / E359D / S406K;

[0447] V59A / D117S / N122D / S124N / N127R / Q128K / E129V / T133E / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0448] V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312D / D318Y / Q319R / T321D / D343K / F353L / E359D / L403F / S406K;

[0449] V59A / D117T / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312E / K317R / D318Y / Q319R / T321D / D343K / F353L / E359D / L403F / S406K;

[0450] V59A / D117W / N122D / S124N / N127L / Q128K / E129V / T133E / Y134F / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312D / D318Y / Q319A / T321D / L322K / D343K / E359D / L403F / S406K;

[0451] V59A / D117M / N122D / S124N / Q128K / E129V / T133E / Q135M / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312D / D318Y / Q319R / T321D / D343K / F353L / E359D / L403F / S406K;

[0452] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0453] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129I / T133E / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282Y / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312D / M316T / D318Y / Q319R / T321D / L322K / D343K / E359D / L403F / S406K;

[0454] V59A / D117E / N122D / S124N / N127L / Q128K / E129V / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282N / M284I / N293Y / K294R / T297E / A304K / T309K / M311L / T312D / D318Y / Q319R / T321D / D343E / F353L / E359D / L403F / S406K;

[0455] V59A / D117T / N122D / S124N / N127R / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / A304K / T309D / M311L / T312K / N313D / D318Y / Q319R / T321D / D343E / F353L / E359D / L403F / S406K;

[0456] V59A / D117T / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / K294R / T297E / A304K / T309N / M311L / T312K / N313D / K317E / D318Y / Q319R / T321E / D343E / F353L / E359D / L403F / S406K;

[0457] V59A / D117S / N122D / S124N / N127L / Q128K / E129K / T133E / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274E / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319H / T321D / D343E / E359D / S406K;

[0458] V59A / D117E / N122D / S124N / Q128K / E129I / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274T / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319H / T321D / D343E / E359D / S406K;

[0459] V59A / D117E / N122D / S124N / Q128K / E129V / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294R / T297E / A304K / T309K / M311L / T312D / N313G / K317R / D318Y / Q319R / T321D / L322K / D343E / F353L / L403F / S406K;

[0460] V59A / D117T / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / K294N / T297K / A304K / T309K / M311L / T312E / N313G / M316T / K317E / D318Y / Q319R / T321D / D343E / F353Y / E359D / L403F / S406K;

[0461] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129V / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312E / D318Y / Q319R / T321D / L322K / D343K / E359D / L403F / S406K;

[0462] V59A / D117T / N122D / S124N / D125N / N127L / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / K294R / T297E / A304K / T309K / M311L / T312D / D318Y / Q319R / T321E / D343E / E359D / L403F / S406K;

[0463] V59A / D117T / N122D / S124Y / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312E / D318Y / Q319R / T321D / D343K / E359D / S406K;

[0464] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129I / T133V / Q135G / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312D / N313D / M316T / D318Y / Q319R / T321D / D343K / E359D / L403F / S406K;

[0465] V59A / D117E / N122D / S124N / Q128K / E129I / T133P / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294R / T297E / A304K / T309K / M311L / T312E / D318Y / Q319R / T321D / D343E / F353L / E359D / L403F / S406K;

[0466] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133E / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282Y / M284I / N293Y / K294N / T297K / A304K / T309K / M311L / T312D / D318Y / Q319R / T321D / L322K / D343E / F353L / E359D / L403F / S406K;

[0467] V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / K294N / T297K / A304K / T309K / M311L / T312E / N313D / D318Y / Q319R / T321D / D343E / F353L / E359D / L403F / S406K;

[0468] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309N / M311L / T312K / N313D / D318Y / Q319R / T321D / L322K / D343K / F353L / E359D / L403F / S406K;

[0469] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / A304K / T309K / M311L / T312D / M316V / D318Y / Q319R / T321D / L322K / D343E / F353Y / E359D / L403F / S406K;

[0470] V59A / D117S / N122D / S124N / D125N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / A304K / T309K / M311E / T312E / M316V / D318Y / Q319R / T321D / D343E / F353Y / E359D / L403F / S406K;

[0471] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129A / I130V / G132D / T133W / Q13 5N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0472] V59A / D117T / N122D / S124N / N127L / Q128K / E129V / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / K294N / T297K / A304K / T309N / M311L / T312K / N313D / M316V / K317E / D318K / Q319H / T321D / D343E / F353Y / E359D / L403F / S406K;

[0473] V59A / D117E / N122D / S124N / N127L / Q128K / E129V / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311E / T312E / M316V / D318Y / Q319R / T321E / D343K / F353Y / E359D / L403F / S406K;

[0474] V59A / D117T / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / K294R / T297E / K300N / A304K / T309D / M311L / T312K / N313D / M316T / K317E / D318Y / Q319R / T321D / D343K / F353Y / E359D / L403F / S406K;

[0475] V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282N / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312E / K317E / D318Y / Q319R / T321D / D343Q / F353L / E359D / L403F / S406K;

[0476] V59A / D117T / N122D / S124N / N127L / Q128K / E129V / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282Y / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312D / M316T / D318Y / Q319R / T321E / L322Y / D343K / F353Y / E359D / L403F / S406K;

[0477] V59A / D117T / N122D / S124N / N127R / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282N / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312D / M316T / K317R / D318Y / Q319R / T321D / D343K / F353L / E359D / L403F / S406K;

[0478] V59A / D117T / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282N / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312E / M316V / K317R / D318Y / Q319R / T321D / D343K / F353Y / E359D / S406K;

[0479] V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282Y / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312D / M316T / D318Y / Q319R / T321D / L322K / D343K / F353Y / E359D / S406K;

[0480] V59A / D117T / N122D / S124N / N127L / Q128K / E129V / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312D / N313G / D318Y / Q319R / T321D / L322K / D343K / F353L / E359D / L403F / S406K;

[0481] V59A / D117E / N122D / S124N / N127L / Q128K / E129V / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282Y / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312D / M316T / D318Y / Q319R / T321D / L322H / D343K / F353Y / E359D / L403F / S406K;

[0482] V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135N / Y176F / K177L / A184E / E188P / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312E / D318Y / Q319R / T321D / D343K / F353L / E359D / L403F / S406K;

[0483] V59A / D117W / N122D / S124N / D125N / N127L / Q128K / E129I / T133E / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282D / M284I / N293Y / K294R / T297E / A304K / T309K / M311L / T312D / D318Y / Q319R / T321D / L322K / D343E / F353L / E359D / L403F / S406K;

[0484] V59A / D117T / N122D / S124N / N127L / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312D / M316V / D318Y / Q319H / T321D / D343K / L403F / S406K;

[0485] V59A / D117T / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282N / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312E / D318Y / Q319R / T321D / D343K / F353L / E359D / L403F / S406K;

[0486] V59A / D117E / N122D / S124N / D125N / N127L / Q128E / E129K / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312D / D318Y / Q319R / T321D / L322K / D343K / F353L / E359D / L403F / S406K;

[0487] V59A / D117T / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / G282Y / M284I / N293Y / K294N / T297K / A304K / T309K / M311L / T312E / D318Y / Q319R / T321D / L322K / D343E / F353L / E359D / L403F / S406K;

[0488] V59A / D117T / N122D / S124N / D125N / N127L / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309K / M311L / T312D / K317R / D318Y / Q319R / T321D / D343Q / E359D / L403F / S406K;

[0489] V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294R / T297E / K300N / A304K / T309K / M311L / T312D / N313D / K317R / D318Y / Q319R / T321D / L322K / D343K / F353L / E359D / L403F / S406K;

[0490] V59A / D117W / N122D / S124N / N127L / Q128K / E129K / T133E / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / I277L / N281D / M284I / N293Y / K294N / T297K / K300N / A304K / T309E / M311E / T312K / N313D / M316V / K317R / D318Y / Q319R / T321D / L322K / D343K / F353Y / E359D / L403F / S406K;

[0491] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / I130V / G132E / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0492] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0493] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / G132D / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0494] V59A / D117E / V121M / N122D / S124N / N127L / Q128K / E129A / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0495] V59A / D117E / N122D / S124N / N127L / Q128K / E129K / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0496] V59A / D117E / V121M / N122D / S124N / D125N / N127L / Q128K / E129A / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0497] V59A / D117E / E120D / N122D / S124N / N127L / Q128K / E129K / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0498] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129V / I130V / G132E / T133W / Y134F / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0499] V59A / D117E / N122D / S124N / N127R / Q128K / E129L / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0500] V59A / D117E / V119Q / N122D / S124N / D125N / N127L / Q128K / E129P / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0501] V59A / D117E / N122D / S124N / N127L / Q128K / E129K / G132E / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0502] V59A / D117E / V119Q / E120A / V121M / N122D / S124N / D125N / N127L / Q128K / E129P / G132E / T133W / Y134E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0503] V59A / D117T / N122D / S124N / N127R / Q128K / E129I / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0504] V59A / D117W / V119Q / N122D / S124N / N127L / Q128K / E129I / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0505] V59A / D117E / N122D / S124N / Q128K / E129I / G132E / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0506] V59A / D117M / V119Y / N122D / S124N / D125N / N127L / Q128K / E129I / I130T / G132D / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0507] V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0508] V59A / D117E / V119Q / N122D / S124N / D125N / N127L / Q128K / E129I / I130V / G132D / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0509] V59A / D117M / N122D / S124N / D125N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0510] V59A / D117E / V119Y / N122D / S124N / N127L / Q128K / E129I / I130V / G132E / T133A / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0511] V59A / D117W / V119Y / E120D / N122D / S124N / Q128K / E129I / G132D / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0512] V59A / D117E / V119Y / N122D / S124N / Q128K / E129I / I130T / G132D / T133A / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0513] V59A / D117E / N122D / S124F / N127R / Q128K / E129I / G132D / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0514] V59A / D117W / V119Y / E120A / N122D / S124N / Q128K / E129I / I130T / G132E / T133E / Y134E / Q135K / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0515] V59A / D117W / E120D / N122D / S124N / N127L / Q128K / E129L / I130V / G132E / T133E / Q13 5E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0516] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132D / T133W / Q135D / Y17 6F / K177L / A184K / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N;

[0517] V59A / D117E / N122D / S124N / Q128K / E129V / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0518] V59A / D117W / N122D / S124N / N127L / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0519] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0520] V59A / D117E / N122D / S124N / Q128K / E129I / T133W / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0521] V59A / D117E / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0522] V59A / D117W / N122D / S124N / N127R / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0523] V59A / D117W / N122D / S124N / Q128K / E129I / T133E / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0524] V59A / D117E / N122D / S124N / Q128K / E129V / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0525] V59A / D117E / N122D / S124N / Q128K / E129I / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0526] V59A / D117E / N122D / S124N / N127L / Q128K / E129K / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0527] V59A / D117E / N122D / S124N / N127R / Q128K / E129V / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0528] V59A / D117E / N122D / S124N / Q128K / E129I / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0529] V59A / D117E / N122D / S124N / Q128K / E129I / T133P / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0530] V59A / D117E / N122D / S124N / Q128K / E129I / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0531] V59A / D117W / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274E / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319H / T321D / D343E / E359D / S406K;

[0532] V59A / D117W / N122D / S124N / Q128K / E129V / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0533] V59A / D117W / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0534] V59A / D117T / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0535] V59A / D117K / N122D / S124N / N127R / Q128K / E129I / T133E / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0536] V59A / D117W / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274T / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319H / T321D / D343E / E359D / S406K;

[0537] V59A / D117E / N122D / S124N / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0538] V59A / D117E / N122D / S124N / Q128K / E129V / T133P / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0539] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0540] V59A / D117E / N122D / S124N / Q128K / E129I / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0541] V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0542] V59A / D117W / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0543] V59A / D117E / N122D / S124N / Q128K / E129I / T133A / Q135T / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / H274K / N281D / M284I / N293Y / T297E / A304K / M311E / T312E / D318E / Q319H / T321D / D343E / E359D / S406K;

[0544] V59A / D117E / N122D / S124N / Q128K / E129I / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311E / T312E / M316T / D318Y / Q319R / T321D / D343E / E359D / S406K;

[0545] V59A / D117T / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311E / T312E / M316L / D318E / Q319R / T321D / D343E / S406K;

[0546] V59A / D117T / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311I / T312E / D318Y / Q319H / T321D / D343E / S406K;

[0547] V59A / D117E / N122D / S124N / N127L / Q128K / E129K / T133V / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311E / T312E / M316T / D318L / Q319H / T321D / D343E / E359D / S406K;

[0548] V59A / D117M / N122D / S124N / Q128K / E129L / T133E / Q135P / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / N281D / M284I / N293Y / T297E / A304K / M311E / T312E / M316L / D318E / Q319H / T321D / D343E / S406K;

[0549] V59A / D117T / N122D / S124N / N127L / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / T297E / A304K / M311F / T312E / D318E / Q319H / T321D / D343E / E359D / S406K;

[0550] V59A / D117S / N122D / S124F / Q128K / E129I / T133E / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274E / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / D318T / Q319H / T321D / D343E / S406K;

[0551] V59A / D117E / N122D / S124N / N127L / Q128K / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311T / T312E / D318E / Q319K / T321D / D343E / E359D / S406K;

[0552] V59A / D117E / N122D / S124N / N127L / Q128K / E129K / T133A / Q135R / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311E / T312E / M316L / D318E / Q319K / T321D / D343E / S406K;

[0553] V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316L / D318E / Q319Y / T321E / D343E / S406K;

[0554] V59A / D117E / N122D / S124N / Q128K / E129I / T133P / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311F / T312E / D318E / Q319K / T321D / D343E / S406K;

[0555] V59A / D117E / N122D / S124F / Q128K / E129I / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316T / D318Y / Q319H / T321D / D343E / E359D / S406K;

[0556] V59A / D117T / N122D / S124N / N127R / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316T / D318E / Q319H / T321D / D343E / S406K; or

[0557] V59A / D117E / N122D / S124N / Q128K / E129I / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q25 4S / H274L / N281D / M284I / N293Y / T297E / A304K / M311F / T312E / D318E / Q319R / T321D / D343E / E359D / S406K.

[0558] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a similar sequence to Bacillus stearothermophilus α-amylase or its mutant, the amino acid alteration may further include substitutions at one or more of the following positions: 117, 122, 124, 128, 129, 130, 132, 133, 135, 176, 177, 191, 212, 274, 277, 281, 304, 311, 312, 318, 319, 321 and / or 406. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of amylases with a sequence similar to that of *Bacillus stearothermophilus* α-amylase or its mutants, the amino acid alteration may further include one or more substitutions selected from the group consisting of: substitution at amino acid position 117: 117E; substitution at amino acid position 122: 122D; substitution at amino acid position 124: 124N; substitution at amino acid position 128: 128K; substitution at amino acid position 129: 129I; substitution at amino acid position 130: 130V; substitution at amino acid position 132: 132E; substitution at amino acid position 133: 133W; substitution at amino acid position 135: 135E; substitution at amino acid position 17... The substitution at position 6 is 176F; the substitution at position 177 is 177L; the substitution at position 191 is 191D; the substitution at position 212 is 212T; the substitution at position 274 is 274E or 274K; the substitution at position 277 is 277L; the substitution at position 281 is 281D; the substitution at position 304 is 304K; the substitution at position 311 is 311L; the substitution at position 312 is 312D, 312E, or 312K; the substitution at position 318 is 318E or 318Y; the substitution at position 319 is 319H or 319Y; the substitution at position 321 is 321D; and the substitution at position 406 is 406K.In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the resulting mutant may further comprise one or more amino acids selected from the group consisting of: amino acid at position 117: 117E; amino acid at position 122: 122D; amino acid at position 124: 124N; amino acid at position 128: 128K; amino acid at position 129: 129I; amino acid at position 130: 130V; amino acid at position 132: 132E; amino acid at position 133: 133W; amino acid at position 135: 135E; amino acid at position 176: 135E; amino acid at position 176: 135E; amino acid at position 135: 135E; amino acid at position 13 ... The amino acid at position 177 is 177L; the amino acid at position 191 is 191D; the amino acid at position 212 is 212T; the amino acid at position 274 is 274E or 274K; the amino acid at position 277 is 277L; the amino acid at position 281 is 281D; the amino acid at position 304 is 304K; the amino acid at position 311 is 311L; the amino acid at position 312 is 312D, 312E, or 312K; the amino acid at position 318 is 318E or 318Y; the amino acid at position 319 is 319H or 319Y; the amino acid at position 321 is 321D; and the amino acid at position 406 is 406K.

[0559] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration of a method for modifying an amylase with a sequence similar to that of *Bacillus stearothermophilus* α-amylase, or a mutant thereof, may further include a substitution at amino acid position 316. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration of a method for modifying an amylase with a sequence similar to that of *Bacillus stearothermophilus* α-amylase, or a mutant thereof, may further include the substitution 316T. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the amino acid alteration of a method for modifying an amylase with a sequence similar to that of *Bacillus stearothermophilus* α-amylase, or a mutant thereof, may further include the amino acid 316T.

[0560] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with a sequence similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the amino acid alteration may further include substitutions at one or more positions of amino acid positions 322, 343, and / or 359. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with a sequence similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the amino acid alteration may further include substitutions selected from the group consisting of 322K, 343E, or 343K, 359D. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with a sequence similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the resulting mutant may further include one or more amino acids selected from the group consisting of 322K, 343E, or 343K, 359D.

[0561] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of an amylase having a sequence similar to that of Bacillus stearothermophilus α-amylase or its mutants, the amino acid alteration may further include substitutions at one or more of the following positions: 282, 296, 300, 305, 309, 313, 317 and / or 403. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the amino acid alteration may further include one or more substitutions selected from the group consisting of: substitution at amino acid position 282 as 282Y; substitution at amino acid position 296 as 296A; substitution at amino acid position 300 as 300N; substitution at amino acid position 305 as 305Y; substitution at amino acid position 309 as 309D or 309K; substitution at amino acid position 313 as 313D; substitution at amino acid position 317 as 317E or 317R; and / or substitution at amino acid position 403 as 403F. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a similar sequence to Bacillus stearothermophilus α-amylase or a mutant thereof, the resulting mutant may further contain one or more amino acids selected from the group consisting of: 282Y, 296A, 300N, 305Y, 309D or 309K, 313D, 317E or 317R, 403F.

[0562] In some embodiments, based on any of the amino acid or amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, the amino acid alteration may further include a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0563] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T321D / S406K;

[0564] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T321D / S406K / M316T / D318Y.

[0565] In some embodiments, based on any of the amino acid or amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to those of Bacillus stearothermophilus α-amylase, the amino acid alteration may further include a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0566] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / N313D / M316T / K317R / D318Y / Q319Y / T321D / L322K / S406K;

[0567] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H27 4K / I277L / N281D / M284I / N293Y / Y296A / T297E / A304K / T309K / M311L / T312D / N313D / M316T / D318Y / Q319Y / T321D / L322K / S406K;

[0568] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / I277L / N28 1D / G282Y / M284I / N293Y / T297E / K300N / A304K / F305Y / T309D / M311L / T 312K / N313D / K317E / D318E / Q319Y / T321D / L322K / D343K / E359D / S406K;

[0569] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274 E / I277L / N281D / M284I / N293Y / Y296A / T297E / A304K / M311L / T312D / M316T / D318Y / Q319H / T321D / L322K / D343E / E359D / S406K / ;

[0570] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / T297E / A304K / F305Y / M311L / T312D / M316T / D318Y / Q319H / T321D / D343E / E359D / S406K; and

[0571] V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / Y296A / T297E / K300N / A304K / M311L / T312D / M316T / D318Y / Q319H / T321D / D343K / E359D / L403F / S406K

[0572] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of Bacillus stearothermophilus α-amylase or its mutants, the amino acid alteration may further include substitutions at one or more of the following positions: 117, 122, 124, 127, 128, 129, 133, 135, 176, 177, 191, 212, 274, 277, 281, 304, 311, 312, 318, 319, 321 and / or 406. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of amylases having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or its mutants, the amino acid alteration may further include one or more substitutions selected from the group consisting of: substitution at amino acid position 117: 117E; substitution at amino acid position 122: 122D; substitution at amino acid position 124: 124N; substitution at amino acid position 127: 127R; substitution at amino acid position 128: 128K; substitution at amino acid position 129: 129I; substitution at amino acid position 133: 133V; substitution at amino acid position 135: 135K; substitution at amino acid position 176: 1... 76F; Substitution at amino acid position 177: 177L; Substitution at amino acid position 191: 191D; Substitution at amino acid position 212: 212T; Substitution at amino acid position 274: 274K or 274E; Substitution at amino acid position 277: 277L; Substitution at amino acid position 281: 281D; Substitution at amino acid position 304: 304K; Substitution at amino acid position 311: 311L; Substitution at amino acid position 312: 312D or 312K; Substitution at amino acid position 318: 318Y or 318E; Substitution at amino acid position 319: 319H or 319Y; Substitution at amino acid position 321: 321D; Substitution at amino acid position 406: 406K.In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the resulting mutant may further comprise one or more amino acids selected from the group consisting of: amino acid at position 117: 117E; amino acid at position 122: 122D; amino acid at position 124: 124N; amino acid at position 127: 127R; amino acid at position 128: 128K; amino acid at position 129: 129I; amino acid at position 133: 133V; amino acid at position 135: 135K; amino acid at position 176: 17... 6F; Amino acid at position 177 is 177L; Amino acid at position 191 is 191D; Amino acid at position 212 is 212T; Amino acid at position 274 is 274K or 274E; Amino acid at position 277 is 277L; Amino acid at position 281 is 281D; Amino acid at position 304 is 304K; Amino acid at position 311 is 311L; Amino acid at position 312 is 312D or 312K; Amino acid at position 318 is 318Y or 318E; Amino acid at position 319 is 319H or 319Y; Amino acid at position 321 is 321D; Amino acid at position 406 is 406K.

[0573] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to *Bacillus stearothermophilus* α-amylase, the amino acid alteration may further include substitutions at one or more positions of amino acid positions 316, 343, and / or 359. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying or mutants of amylases with sequences similar to *Bacillus stearothermophilus* α-amylase, the amino acid alteration may further include substitutions selected from the group consisting of: a substitution at amino acid position 316 of 316T; a substitution at amino acid position 343 of 343 of 343E or 343K; and / or a substitution at amino acid position 359 of 359D. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of Bacillus stearothermophilus α-amylase or a mutant thereof, the resulting mutant may further contain one or more amino acids selected from the group consisting of: amino acid at amino acid position 316 being 316T; amino acid at amino acid position 343 being 343E or 343K; and / or amino acid at amino acid position 359 being 359D.

[0574] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the amino acid alteration may further include substitutions at amino acid positions 296 and / or 322. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the amino acid alteration may further include one or more substitutions selected from the group consisting of: 296A, 322K. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or mutants thereof, the resulting mutant may further include one or more amino acids selected from the group consisting of: 296A, 322K.

[0575] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of an amylase having a sequence similar to that of Bacillus stearothermophilus α-amylase or a mutant thereof, the amino acid alteration may further include substitutions at one or more of the following positions: amino acid positions 282, 300, 305, 309, 313, 317 and / or 403. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, for the modification of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the amino acid alteration may further include one or more substitutions selected from the group consisting of: substitution at amino acid position 282 as 282Y; substitution at amino acid position 300 as 300N; substitution at amino acid position 305 as 305Y; substitution at amino acid position 309 as 309D or 309K; substitution at amino acid position 313 as 313D; substitution at amino acid position 317 as 317E; and / or substitution at amino acid position 403 as 403F. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification method of an amylase having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or a mutant thereof, the resulting mutant may further include one or more amino acids selected from the group consisting of: amino acid at position 282: 282Y; amino acid at position 300: 300N; amino acid at position 305: 305Y; amino acid at position 309: 309D or 309K; amino acid at position 313: 313D; amino acid at position 317: 317E; and / or amino acid at position 403: 403F.

[0576] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification of amylases having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or mutants thereof, the resulting mutants may contain a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0577] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E18 8P / T191D / V212T / Q254S / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T321D / S406K;

[0578] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V21 2T / Q254S / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T321D / S406K / T312D / M316T / D318Y / E359D.

[0579] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification of amylases having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or mutants thereof, the resulting mutants may contain a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0580] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / Y296A / T297E / A304K / T309K / M311L / T312D / N313D / M316T / D318Y / Q319Y / T321D / L322K / S406K;

[0581] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / I277L / N281D / G282Y / M284I / N293Y / T297E / K300N / A304K / F305Y / T309D / M311L / T312K / N313D / K317E / D318E / Q319Y / T321D / L322K / D343K / E359D / S406K;

[0582] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / Y296A / T297E / A304K / M311L / T312D / M316T / D318Y / Q319H / T321D / L322K / D343E / E359D / S406K;

[0583] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / T297E / A304K / F305Y / M311L / T312D / M316T / D318Y / Q319H / T321D / D343E / E359D / S406K; and

[0584] V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / I27 7L / N281D / M284I / N293Y / Y296A / T297E / K300N / A304K / M311L / T312D / M316T / D318Y / Q319H / T321D / D343K / E359D / L403F / S406K.

[0585] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for the modification of amylases having a sequence similar to that of *Bacillus stearothermophilus* α-amylase or mutants thereof, the resulting mutants may contain a group of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO:5:

[0586] D117K / N122D / S124N / Q128K / T133E / Q135E / Q137A;

[0587] N122D / S124N / Q128K / T133V / Q135K / Q137A;

[0588] D117T / N122D / S124A / Q128R / T133E / Y134H / Q135R / Q137K;

[0589] A302K / M309L / T310D / M314V / K315R / Q317Y; and

[0590] A302G / M309L / T310N / N311G / M314V / K315S / Q317H.

[0591] Based on any of the amino acids or amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or their mutants, the amino acid changes in the parent or mutant may further include one or more amino acid changes selected from the group consisting of: 180*, 181*, 193F, and 416G. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or their mutants, the amino acid changes in the parent or mutant may further include the following amino acid changes: 180* / 181* / 193F / 416G. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, for methods of modifying amylases with sequences similar to *Bacillus stearothermophilus* α-amylase or their mutants, the amino acid changes in the parent or mutant may further include the following amino acid changes: G180* / I181* / N193F / V416G.

[0592] B. Methods for modifying amylases with sequences similar to Bacillus licheniformis α-amylase (L-type amylase) and their mutants.

[0593] In some embodiments, the parent enzyme (or an α-amylase with a similar three-dimensional structure to the reference *Bacillus stearothermophilus* α-amylase) is an α-amylase with a similar sequence to *Bacillus licheniformis* α-amylase. For example, it may comprise the amino acid sequence shown in any one of SEQ ID NO: 25-39, or have a sequence identity of 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%, at least 99%, and less than 100% with the amino acid sequence shown in any one of SEQ ID NO: 25-39.

[0594] In some embodiments, mutants of amylases having a sequence similar to that of Bacillus licheniformis α-amylase contain amino acid alterations relative to the parental α-amylase, which will be described in detail below.

[0595] In some embodiments, the parental α-amylase may be the amino acid sequence shown in SEQ ID NO:26 (referred to herein as mutant M35), which has the amino acid alterations V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W relative to the α-amylase mutant LE399, and has improved α-amylase activity, thermostability, and / or low pH stability relative to mutant LE399. In some embodiments, the parental α-amylase may be wild-type BLA (SEQ ID NO:25). In some embodiments, the parental α-amylase may be mutant LE399 (SEQ ID NO:39). In some embodiments, the parental α-amylase may be any one of the α-amylases in SEQ ID NO:27-38. In some embodiments, the parental α-amylase may be a mature polypeptide (e.g., a mature polypeptide of M35, wild-type BLA, or LE399) that does not contain a signal peptide. In some embodiments, the parental α-amylase may be a mature polypeptide as described in any of SEQ ID NO:25-39. In some embodiments, the parental α-amylase contains a signal peptide (e.g., M35, wild-type BLA, or LE399 containing a signal peptide, such as a polypeptide obtained by adding a signal peptide to the N-terminus of SEQ ID NO:26, SEQ ID NO:25, or SEQ ID NO:39). In some embodiments, the parental α-amylase is a polypeptide obtained by adding a signal peptide to the N-terminus of any of the polypeptides described in SEQ ID NO:27-38.

[0596] In this article, for amylases or their mutants that have similar sequences to Bacillus licheniformis α-amylase, unless otherwise specified, the positions of amino acids in the described α-amylase mutants are determined based on the amino acid sequence shown in SEQ ID NO:25, a reference amino acid sequence.

[0597] In some embodiments, for the modification method of an amylase having a sequence similar to that of Bacillus licheniformis α-amylase or a mutant thereof, region 3 includes amino acids at positions 116-136, region 4 includes amino acids at positions 175-195, and region 6 includes amino acids at positions 296-320; wherein the amino acid position number corresponds to the position number of SEQ ID NO:25(BLA).

[0598] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, amino acid changes within region 3 include:

[0599] (1) Amino acid substitutions at any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions from amino acid positions 116, 118, 123, 124, 126, 127, 128, 131, 132, 133, 134 and 136, preferably amino acid substitutions at 5-10 positions, 5-7 positions or 6-10 positions;

[0600] (2) Amino acid substitutions at any one, two, three, four, five, six, or seven positions from amino acid positions 116, 123, 126, 127, 128, 132, and 134, preferably at five to seven positions; or

[0601] (3) Amino acid substitutions at any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions from amino acid positions 116, 123, 124, 126, 127, 128, 132, 133, 134 and 136, preferably amino acid substitutions at 5-10 positions, 5-7 positions or 6-10 positions;

[0602] The amino acid position number therein corresponds to the position number of SEQ ID NO:25(BLA).

[0603] The specific amino acid substitutions at the aforementioned positions are described below.

[0604] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid substitutions within region 3 include one or more of the substitutions described below:

[0605] The amino acid at position 116 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, Q, E, R, D, M, K, W or L, more preferably by E, R, D, M, K, W or L, or by D, E, K, L, M, N or Q, or by D, E, K, L, M, N, Q or R.

[0606] The amino acid position 123 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by E, H, I, K, Q, R, V, N, F, Y, L or T, more preferably by N, F, Y, L or T, or by E, H, I, K, N, Q, R, T, V or Y, or by E, F, H, I, K, N, L, Q, R, T, V or Y;

[0607] The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably N;

[0608] The amino acid at position 126 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L or C, more preferably C;

[0609] The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by H, Q, E, R, D, M, K, W or L, more preferably by E, R, D, M, K, W or L or by E, H, K or Q;

[0610] The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by M, Q, R, T, P, L, K, I, E, D or A, more preferably by P, L, K, I, E, D or A or by A, D, E, I, K, M, P, Q, R or T;

[0611] The amino acid at position 131 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D, E or K;

[0612] The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V. Preferably, it is replaced by D, T, V, W, P, A, or K. More preferably, it is replaced by W, P, A, or K, or by D, K, P, T, or V, or by D, K, P, T, V, or W.

[0613] The amino acid at position 133 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably F or Y, more preferably Y;

[0614] The amino acid at position 134 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V; preferably, it is substituted with A, D, E, F, K, M, P, N, Q, S, T, V, or W; more preferably, it is substituted with K, W, M, T, E, P, N, D, or A, or with A, E, F, K, M, P, Q, S, T, V, or W; and

[0615] The amino acid at position 136 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably A, D, or E, and preferably A.

[0616] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, amino acid changes within region 4 include:

[0617] (1) Amino acid substitutions at any one, two, three, four, or five positions from amino acid positions 175, 178, 181, 188, and 191, preferably at two to four or three to four positions; or

[0618] (2) Amino acid substitution at any one, two, three or four positions in amino acid positions 175, 181, 188 and 191, preferably at two to four positions or three to four positions;

[0619] The amino acid position number corresponds to the position number of SEQ ID NO:25(BLA).

[0620] The specific amino acid substitutions at the above positions are described below.

[0621] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid substitutions within region 4 include one or more of the substitutions described below:

[0622] The amino acid at position 175 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably F or W, more preferably F.

[0623] The amino acid at position 178 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or E, more preferably K.

[0624] The amino acid at position 181 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by T, A, D, E, G, N, R or S, more preferably by E.

[0625] The amino acid at position 188 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with D, E, G, K, N, R, S, or T, more preferably with T, R, K, or D, or with E, G, K, N, S, or T; and

[0626] The amino acid at position 191 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably C.

[0627] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, amino acid changes within region 6 include:

[0628] (1) Amino acid substitutions at any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 positions of amino acid positions 298, 301, 302, 306, 307, 308, 309, 312, 313, 314, 315, 316, 318, 319 and 320, preferably amino acid substitutions at 5-8 positions or 6-7 positions;

[0629] (2) Amino acid substitutions at any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions of amino acid positions 298, 301, 302, 307, 309, 312, 314, 315, 316, 318, 319 and 320, preferably amino acid substitutions at 5-8 positions or 6-7 positions;

[0630] (3) Amino acid substitutions at any 1, 2, 3, 4, 5, 6, 7, 8 or 9 positions in amino acid positions 301, 302, 306, 308, 309, 313, 316, 318 and 320, preferably at 5-8 positions or 6-7 positions;

[0631] (4) Amino acid substitutions at any 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions from amino acid positions 301, 302, 306, 308, 309, 313, 315, 316, 318 and 320, preferably at 5-8 positions or 6-7 positions;

[0632] (5) Amino acid substitution at any one, two, three, four, five, six or seven positions in amino acid positions 301, 302, 308, 309, 316, 318 and 320, preferably at six or seven positions;

[0633] (6) Or amino acid substitution at any one, two, three, four, five, six, seven or eight positions in amino acid positions 301, 302, 308, 309, 315, 316, 318 and 320, preferably amino acid substitution at five to eight positions or six to seven positions;

[0634] (7) Substitution of amino acids at any one, two, three, four, five, or six positions from amino acid positions 301, 302, 309, 316, 318, and 320, or

[0635] (8) Amino acid substitutions at any one, two, three, four, five, six or seven positions in amino acid positions 301, 302, 309, 315, 316, 318 and 320, preferably amino acid substitutions at six or seven positions;

[0636] The amino acid position number corresponds to the position number of SEQ ID NO:25(BLA).

[0637] The specific amino acid substitutions at the above positions are described below.

[0638] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid substitutions within region 6 include one or more of the substitutions described below:

[0639] The amino acid at position 298 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A or S.

[0640] The amino acid at position 301 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, E, H, K, L, N, Q, R, S, T, V or Y, more preferably K;

[0641] The amino acid at position 302 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, F, G, L, V or W, more preferably F.

[0642] The amino acid at position 306 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably H, R or Y.

[0643] The amino acid at position 307 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L or I.

[0644] The amino acid at position 308 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by F or M;

[0645] The amino acid at position 309 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by R, A, D, E, H or Q, more preferably by E or D.

[0646] The amino acid at position 313 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L;

[0647] The amino acid at position 314 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A;

[0648] The amino acid at position 315 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably E or T or V, more preferably T or V.

[0649] The amino acid position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, E, F, L, M, Q, R, V, W, Y or E, more preferably Y.

[0650] The amino acid at position 318 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with D, E, or Q, more preferably with E or D; and

[0651] The amino acid at position 320 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably A.

[0652] In some implementations, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, around region 6 Amino acid changes within the range include:

[0653] (1) Amino acid substitutions at any one, two, three, four, five, six, or seven positions from amino acid positions 271, 274, 281, 294, 340, 356, and 406, preferably at positions 4-7, 5-7, or 4-6; or

[0654] (2) Amino acid substitution at any one, two, three, four, five or six positions in amino acid positions 271, 281, 294, 340, 356 and 406, preferably at four to six positions;

[0655] The amino acid position number corresponds to the position number of SEQ ID NO:25(BLA).

[0656] The specific amino acid substitutions at the above positions are described below.

[0657] In some implementations, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, around region 6 The amino acid substitutions within the range are selected from one or more of the following substitutions:

[0658] The amino acid at position 271 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or L, more preferably L;

[0659] The amino acid at position 274 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by I, M or V.

[0660] The amino acid at position 281 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by C, F, I, L, M, T or V, more preferably by M.

[0661] The amino acid at position 294 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D, E, K, L, N, Q, R, S, T, V or Y, more preferably by Q, N or E.

[0662] The amino acid at position 340 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, D, E, K, M, N, P or R, more preferably A, E or P.

[0663] The amino acid at position 356 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E or D; and

[0664] The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably A, D, K, P, or R, and more preferably K.

[0665] In some embodiments, the modification method for amylases with sequences similar to Bacillus licheniformis α-amylase, or its mutants, includes, in addition to the regions 3, 4, 6, and around region 6 mentioned above. In addition to amino acid changes in any one, two, three, or all four regions within the range, the mutant also includes amino acid substitutions at any one, two, three, or all four positions selected from the following amino acid positions: 156, 205, 265, and 278.

[0666] In some embodiments, amino acid position 156 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with H, T, or F, more preferably with H or T or with F or H.

[0667] In some embodiments, the amino acid position 205 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by D or N, and more preferably by D.

[0668] In some embodiments, amino acid position 265 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with G.

[0669] In some embodiments, amino acid position 278 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with D, E, G, or K, and more preferably with K.

[0670] In some embodiments, the method for modifying an amylase having a sequence similar to that of Bacillus licheniformis α-amylase, or a mutant thereof, includes regions 3, 4, 6, and around region 6 mentioned above. Amino acid substitutions within the range, of which

[0671] The amino acid substitutions in region 3 include at least the amino acid substitutions at amino acid positions 116, 123, 126, 127, 128, 132 and 134;

[0672] The amino acid substitutions in region 4 include at least the amino acid substitutions at amino acid positions 181, 188, and 191;

[0673] The amino acid substitutions within region 6 include at least the amino acid substitutions at positions 301, 302, 308, 309, 316, 318, and 320; and

[0674] Area 6 and surrounding The amino acid substitutions within the range include at least the amino acid substitutions at positions 271, 281, 294, 340, 356, and 406;

[0675] The position numbers of each amino acid correspond to the position numbers of SEQ ID NO:25(BLA), and the amino acid substitutions at each position are described above.

[0676] In some embodiments, the method for modifying an amylase having a sequence similar to that of Bacillus licheniformis α-amylase, or a mutant thereof, includes regions 3, 4, 6, and around region 6 mentioned above. Amino acid substitutions within the range, of which

[0677] The amino acid substitutions in region 3 include at least the amino acid substitutions at amino acid positions 116, 123, 126, 127, 128, 132 and 134;

[0678] The amino acid substitutions in region 4 include at least the amino acid substitutions at amino acid positions 181, 188, and 191;

[0679] The amino acid substitutions within region 6 include at least the amino acid substitutions at positions 301, 302, 309, 316, 318, and 320; and

[0680] Area 6 and surrounding The amino acid substitutions within the range include at least the amino acid substitutions at positions 271, 281, 294, 340, 356, and 406;

[0681] The position numbers of each amino acid correspond to the position numbers of SEQ ID NO:25(BLA), and the amino acid substitutions at each position are described above.

[0682] In some embodiments, the method for modifying an amylase having a sequence similar to that of Bacillus licheniformis α-amylase, or a mutant thereof, includes regions 3, 4, 6, and around region 6 mentioned above. Amino acid substitutions within the range, of which

[0683] The amino acid substitutions in region 3 include at least the amino acid substitutions at amino acid positions 116, 123, 126, 127, 128, 132 and 134;

[0684] The amino acid substitutions in region 4 include at least the amino acid substitutions at amino acid positions 181 and 191;

[0685] The amino acid substitutions within region 6 include at least the amino acid substitutions at positions 301, 309, 316, 318, and 320; and

[0686] Area 6 and surrounding The amino acid substitutions within the range include at least the amino acid substitutions at positions 271, 281, 294, 340, 356, and 406;

[0687] The position numbers of each amino acid correspond to the position numbers of SEQ ID NO:25(BLA), and the amino acid substitutions at each position are described above.

[0688] In some embodiments, the modification method for amylases with sequences similar to Bacillus licheniformis α-amylase, or its mutants, includes, in addition to the regions 3, 4, 6, and around region 6 mentioned above. In addition to amino acid changes in any one, two, three, or all four regions within the range (or optionally further include amino acid substitutions at one or more of the amino acid positions 156, 205, 265, and 278), the mutant also includes amino acid substitutions at one or more positions selected from the following amino acid positions: 23, 39, 49, 61, 62, 74, 106, 114, 148, 154, 155, 172, 212, 213, 242, 276, 370, 407, 417, and 456.

[0689] In some embodiments, the modification method for amylases with sequences similar to Bacillus licheniformis α-amylase, or its mutants, includes, in addition to the regions 3, 4, 6, and around region 6 mentioned above. In addition to amino acid changes in any one, two, three, or all four regions within the range (or optionally further include amino acid substitutions at one or more of the amino acid positions 156, 205, 265, and 278), the mutant also includes amino acid substitutions at one or more of the following amino acid positions: 23, 39, 49, 61, 62, 74, 106, 114, 140, 142, 148, 152, 154, 155, 172, 212, 213, 242, 276, 370, 407, 417, and 456.

[0690] In some embodiments, amino acid position 23 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably with M, Q or S.

[0691] In some embodiments, amino acid position 39 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with C or T.

[0692] In some embodiments, amino acid position 49 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by A, V, or Y.

[0693] In some embodiments, the amino acid position 61 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by M.

[0694] In some embodiments, amino acid position 62 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with W.

[0695] In some embodiments, amino acid position 74 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with A.

[0696] In some embodiments, amino acid position 106 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with L.

[0697] In some embodiments, amino acid position 114 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with F, L, or M.

[0698] In some embodiments, the amino acid position 140 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by K or R, and more preferably by R.

[0699] In some embodiments, amino acid position 142 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with D.

[0700] In some embodiments, amino acid position 148 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D or T or N, more preferably by D or N, and even more preferably by N.

[0701] In some embodiments, amino acid position 152 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably with S.

[0702] In some embodiments, amino acid position 154 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by L, R, T, or Y.

[0703] In some embodiments, the amino acid position 155 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y.

[0704] In some embodiments, amino acid position 172 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E, K, L, or W.

[0705] In some embodiments, the amino acid position 212 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with L.

[0706] In some embodiments, the amino acid position 213 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with K.

[0707] In some embodiments, amino acid position 242 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with A, E, F, L, N, S, or T.

[0708] In some embodiments, amino acid position 276 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with S.

[0709] In some embodiments, amino acid position 370 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with H, Q, or R.

[0710] In some embodiments, amino acid position 407 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E or R.

[0711] In some embodiments, amino acid position 417 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with P.

[0712] In some embodiments, amino acid position 456 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with L or V.

[0713] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid changes include amino acid positions 48, 49, 52, 68, 101, 111, 114, 116, 118, 123, 124, 126, 127, 128, 131, 132, 133, 134, 136, 140, 142, 148, 152, 156, 167, 168, 169, 170, 171, 172, 173, The mutant has substitutions at one or more of the following positions: 175, 178, 181, 188, 191, 205, 265, 271, 272, 275, 278, 279, 280, 281, 283, 291, 294, 298, 301, 302, 307, 309, 312, 314, 315, 316, 318, 319, 320, 338, 340, 356, 372, 373, 374, 375, and 406, wherein the amino acid positions correspond to the positions in SEQ ID NO:25, wherein the mutant has at least 60% and less than 100% sequence identity with the amino acid sequence or mature polypeptide of any of SEQ ID NO:25-39, and wherein the mutant has α-amylase activity.

[0714] In some embodiments, the obtained mutant has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO:26, for example, at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99%, but less than 100%. In some embodiments, the obtained mutant is sequence-aligned with the amino acid sequence shown in SEQ ID NO:26 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide).

[0715] In some embodiments, the obtained mutant has at least 60% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:25-39, for example, at least 60%, at least 65%, at least 70%, at least 75%, 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%, or at least 99%, but less than 100%. In some embodiments, the obtained mutant is sequence-aligned with the amino acid sequence shown in any one of SEQ ID NO:25-39 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide).

[0716] In some embodiments, the mutant of the amylase having a sequence similar to that of Bacillus licheniformis α-amylase is a mature polypeptide that does not contain a signal peptide and / or a leader sequence. In some embodiments, the mutant of the amylase having a sequence similar to that of Bacillus licheniformis α-amylase contains a signal peptide and / or a leader sequence. In some embodiments, the signal peptide may have an amino acid sequence as shown in SEQ ID NO:42.

[0717] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, the amino acid changes include substitutions at one or more of the following positions:

[0718] The amino acid at position 48 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S;

[0719] The amino acid at position 49 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A, V or Y, more preferably A;

[0720] The amino acid at position 52 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or S;

[0721] The amino acid at position 68 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0722] The amino acid at position 101 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0723] The amino acid at position 111 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S;

[0724] The amino acid at position 114 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by F, L, M, E or T, more preferably by E or T or by F, L or M.

[0725] The amino acid at position 116 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by N or Q or D or E or K or L or M or R or W, more preferably by E, R, D, M, K, W or L or D, E, K, L, M, N or Q or D, E, K, L, M, N, Q or R;

[0726] The amino acid at position 118 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably by Q;

[0727] The amino acid position 123 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V. Preferably, it is replaced by E or H or I or K or Q or R or V or F or L or N or T or Y. More preferably, it is replaced by N, F, Y, L or T or by E, H, I, K, N, Q, R, T, V or Y or by E, F, H, I, K, N, L, Q, R, T, V or Y.

[0728] The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0729] The amino acid at position 126 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C or L, more preferably C.

[0730] The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by H or Q or E or R or D or M or K or W or L, more preferably by E, R, D, M, K, W or L or by E, H, K or Q, more preferably by E or K;

[0731] The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. Preferably, it is replaced by M, Q, R, T, A, D, E, I, K, L, or P. More preferably, it is replaced by P, L, K, I, E, D, or A, or by A, D, E, I, K, M, P, Q, R, or T, or by A, D, E, I, K, L, or P.

[0732] The amino acid at position 131 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D, E or K.

[0733] The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or T or V or A or K or P or W, more preferably by W, P, A or K, or by D, K, P, T or V or by D, K, P, T, V or W;

[0734] The amino acid at position 133 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F or Y, more preferably Y;

[0735] The amino acid position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or F or D or E or K or M or N or P or T or W, more preferably D or E or K or M or N or P or T or W.

[0736] The amino acid at position 136 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A, D or E, more preferably A;

[0737] The amino acid at position 140 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or R, more preferably R;

[0738] The amino acid at position 142 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably D;

[0739] The amino acid at position 148 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or T or N, more preferably by D or N or D or T, and even more preferably by N.

[0740] The amino acid at position 152 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S;

[0741] The amino acid at position 156 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by H or T or F, more preferably by H or T or by F or H.

[0742] The amino acid at position 167 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0743] The amino acid at position 168 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K;

[0744] The amino acid at position 169 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably T;

[0745] The amino acid at position 170 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0746] The amino acid at position 171 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K;

[0747] The amino acid at position 172 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by E, K or L or W, more preferably by K.

[0748] The amino acid at position 173 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0749] The amino acid at position 175 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F or W, more preferably F;

[0750] The amino acid at position 178 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or K, more preferably K;

[0751] The amino acid at position 181 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by T or A or D or E or G or N or R or S, more preferably by E.

[0752] The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by E or G or N or S or D or K or R or T, more preferably by T, R, K or D or by E, G, K, N, S or T.

[0753] The amino acid at position 191 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C;

[0754] The amino acid at position 205 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or N, more preferably by D;

[0755] The amino acid at position 265 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably G;

[0756] The amino acid at position 271 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or L, more preferably L;

[0757] The amino acid at position 272 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R;

[0758] The amino acid at position 275 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E;

[0759] The amino acid at position 278 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E or G or K, more preferably by K;

[0760] The amino acid at position 279 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0761] The amino acid at position 280 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V;

[0762] The amino acid at position 281 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C or F or I or L or T or V or M, more preferably M.

[0763] The amino acid at position 283 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0764] The amino acid at position 291 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or R.

[0765] The amino acid position 294 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E or K or L or N or Q or R or S or T or V or Y, more preferably by Q, N or E.

[0766] The amino acid at position 298 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or S.

[0767] The amino acid position 301 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or E or H or L or N or Q or R or S or T or V or Y or K, more preferably K.

[0768] The amino acid at position 302 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. Preferably, it is replaced by A, G, L, V, W, or F. More preferably, it is replaced by F.

[0769] The amino acid at position 307 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L or I.

[0770] The amino acid at position 309 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R or A or H or Q or D or E, more preferably by E or D.

[0771] The amino acid at position 312 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0772] The amino acid at position 314 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0773] The amino acid at position 315 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or T or V, more preferably T or V.

[0774] The amino acid position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or E or F or L or M or Q or R or V or W or E or Y, more preferably Y.

[0775] The amino acid position 318 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by Q or D or E, more preferably by E or D.

[0776] The amino acid at position 319 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0777] The amino acid at position 320 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0778] The amino acid at position 338 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably P, Q or R.

[0779] The amino acid at position 340 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or K or M or N or R or A or E or P, more preferably by A, E or P.

[0780] The amino acid at position 356 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E;

[0781] The amino acid at position 372 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably P or S.

[0782] The amino acid at position 373 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by G, L or T.

[0783] The amino acid at position 374 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or S.

[0784] The amino acid at position 375 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or K or N or T.

[0785] The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or D or P or R or K, more preferably K.

[0786] The amino acid position thereon corresponds to the position in SEQ ID NO:25.

[0787] In some embodiments, the method for modifying an amylase having a sequence similar to that of Bacillus licheniformis α-amylase, or a mutant thereof, yields a mutant containing one or more amino acids selected from the group consisting of: 48S, 49A, 49V, 49Y, 52D, 52S, 68N, 101L, 111S, 114F, 114L, 114M, 114E, 114T, 116N, 116Q, 116D, 116E, 116K, 116L, 116M, 116R, 116W, 118Q, 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123F, 123L, 123N, 123T, 123Y, 124N, 1 26C, 126L, 127E, 127K, 128M, 128Q, 128R, 128T, 128A, 128D, 128E, 128I, 128 K, 128L, 128P, 131D, 131E, 131K, 132D, 132T, 132V, 132A, 132K, 132P, 132W, 1 33F, 133Y, 134A, 134F, 134Q, 134S, 134V, 134D, 134E, 134K, 134M, 134N, 134 P, 134T, 134W, 136A, 136D, 136E, 140K, 140R, 142D, 148D, 148T, 148N, 152S, 1 56H, 156T, 156F, 167Y, 168K, 169T, 170N, 171K, 172E, 172L, 172W, 172K, 173 A, 175F, 175W, 178E, 178K, 181T, 181A, 181D, 181G, 181N, 181R, 181S, 181E, 188E, 188G, 188N, 188S, 188D, 188K, 188R, 188T, 191C, 205D, 205N, 265G, 27 1K, 271L, 272R, 275E, 278D, 278E, 278G, 278K, 279Y, 280V, 281C, 281F, 281I, 281L, 281T, 281V, 281M, 283L, 291K, 291R, 294D, 294L, 294R, 294S, 294T, 29 4V, 294Y, 294E, 294K, 294N, 294Q, 298A, 298S, 301A, 301E, 301H, 301L, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y, 301K, 302A, 302G, 302L, 302V, 302W, 30 2F, 307L, 307I, 309R, 309A, 309H, 309Q, 309D, 309E, 312L, 314A, 315E, 315T,315V, 316A, 316E, 316F, 316L, 316M, 316Q, 316R, 316V, 316W, 316E, 316Y, 318Q, 318D, 318E, 319L, 320A, 338P, 338Q, 338R, 340D, 340K, 340M, 340N, 340R, 340A, 340E, 340P, 356D, 356E, 372P, 372S, 373G, 373L, 373T, 374D, 374S, 375D, 375K, 375N, 375T, 406A, 406D, 406P, 406R, and 406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:25. ,

[0788] In some embodiments, the method for modifying an amylase having a sequence similar to that of Bacillus licheniformis α-amylase, or a mutant thereof, yields a mutant containing one or more amino acids selected from the group consisting of: 48S, 49A, 52D, 52S, 68N, 101L, 111S, 114E, 114T, 116D, 116E, 116K, 116L, 116M, 116R, 116W, 118Q, 123F, 123L, 123N, 123T, 123Y, 124N, 126C, 126 L, 127E, 127K, 128A, 128D, 128E, 128I, 128K, 128L, 128P, 131D, 131E, 131K, 132A, 132K, 132P, 132W, 133F, 133Y, 13 4D, 134E, 134K, 134M, 134N, 134P, 134T, 134W, 136A, 136D, 136E, 140K, 140R, 142D, 148D, 148T, 152S, 156H, 156T, 16 7Y, 168K, 169T, 170N, 171K, 172K, 173A, 175F, 178E, 178K, 181E, 188D, 188K, 188R, 188T, 191C, 205D, 265G, 271K, 2 71L, 272R, 275E, 278D, 278E, 278G, 278K, 279Y, 280V, 281M, 283L, 291K, 291R, 294E, 294K, 294N, 294Q, 298A, 298S, 3 01K, 302F, 307I, 309D, 309E, 312L, 314A, 315E, 315T, 315V, 316Y, 318D, 318E, 319L, 320A, 338P, 338Q, 338R, 340A, 340E, 340P, 356D, 356E, 372P, 372S, 373G, 373L, 373T, 374D, 374S, 375D, 375K, 375N, 375T, 406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:25.

[0789] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid alterations include one or more substitutions selected from the group consisting of: 48S, 49A, 49V, 49Y, 52D, 52S, 68N, 101L, 111S, 114F, 114L, 114M, 114E, 114T, 116N, 116Q, 116D, 116E, 116K, 116L, 116M, 116R, 116W, 118Q, 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123F, 123L, 123N, 123T, 123Y, 124N, 126C , 126L, 127E, 127K, 128M, 128Q, 128R, 128T, 128A, 128D, 128E, 128I, 128K, 1 28L, 128P, 131D, 131E, 131K, 132D, 132T, 132V, 132A, 132K, 132P, 132W, 133F ,133Y, 134A, 134F, 134Q, 134S, 134V, 134D, 134E, 134K, 134M, 134N, 134P, 1 34T, 134W, 136A, 136D, 136E, 140K, 140R, 142D, 148D, 148T, 148N, 152S, 156H ,156T, 156F, 167Y, 168K, 169T, 170N, 171K, 172E, 172L, 172W, 172K, 173A, 1 75F, 175W, 178E, 178K, 181T, 181A, 181D, 181G, 181N, 181R, 181S, 181E, 188E , 188G, 188N, 188S, 188D, 188K, 188R, 188T, 191C, 205D, 205N, 265G, 271K, 2 71L, 272R, 275E, 278D, 278E, 278G, 278K, 279Y, 280V, 281C, 281F, 281I, 281L , 281T, 281V, 281M, 283L, 291K, 291R, 294D, 294L, 294R, 294S, 294T, 294V, 2 94Y, 294E, 294K, 294N, 294Q, 298A, 298S, 301A, 301E, 301H, 301L, 301N, 301Q , 301R, 301S, 301T, 301V, 301Y, 301K, 302A, 302G, 302L, 302V, 302W, 302F, 30 7L, 307I, 309R, 309A, 309H, 309Q, 309D, 309E, 312L, 314A, 315E, 315T, 315V,316A, 316E, 316F, 316L, 316M, 316Q, 316R, 316V, 316W, 316E, 316Y, 318Q, 318D, 318E, 319L, 320A, 338P, 338Q, 338R, 340D, 340K, 340M, 340N, 340R, 340A, 340E, 340P, 356D, 356E, 372P, 372S, 373G, 373L, 373T, 374D, 374S, 375D, 375K, 375N, 375T, 406A, 406D, 406P, 406R, and 406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:25.

[0790] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid alterations include one or more substitutions selected from the group consisting of: 48S, 49A, 52D, 52S, 68N, 101L, 111S, 114E, 114T, 116D, 116E, 116K, 116L, 116M, 116R, 116W, 118Q, 123F, 123L, 123N, 123T, 123Y, 124N, 126C, 126L, 127E, 127K, 128A, 128D, 128E, 128I, 128K, 128L, 128P, 131D, 131E, 131K, 132A, 132K, 132P, 132W, 133F, 133Y, 134D , 134E, 134K, 134M, 134N, 134P, 134T, 134W, 136A, 136D, 136E, 140K, 140R, 142D, 148D, 148T, 152S, 156H, 156T, 167 Y, 168K, 169T, 170N, 171K, 172K, 173A, 175F, 178E, 178K, 181E, 188D, 188K, 188R, 188T, 191C, 205D, 265G, 271K, 27 1L, 272R, 275E, 278D, 278E, 278G, 278K, 279Y, 280V, 281M, 283L, 291K, 291R, 294E, 294K, 294N, 294Q, 298A, 298S, 3 01K, 302F, 307I, 309D, 309E, 312L, 314A, 315E, 315T, 315V, 316Y, 318D, 318E, 319L, 320A, 338P, 338Q, 338R, 340A, 340E, 340P, 356D, 356E, 372P, 372S, 373G, 373L, 373T, 374D, 374S, 375D, 375K, 375N, 375T, 406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:25.

[0791] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid alterations include one or more substitutions selected from the group consisting of: A48S, I49A, I49V, I49Y, A52D, A52S, W68N, V101L, A111S, W114F, W114L, W114M, W114E, W114T, T116N, T116Q, T116D, T116E, T116K, T116L, T116M, T116R, T116W, V118Q, A123E, A123H, A123I, A123K, A123Q, A123R, A123V, A1 23F, A123L, A123N, A123T, A123Y, D124N, N126C, N126L, R127E, R127K, V128 M, V128Q, V128R, V128T, V128A, V128D, V128E, V128I, V128K, V128L, V128P, G131D, G131E, G131K, E132D, E132T, E132V, E132A, E132K, E132P, E132W, H1 33F, H133Y, R134A, R134F, R134Q, R134S, R134V, R134D, R134E, R134K, R134M , R134N, R134P, R134T, R134W, K136A, K136D, K136E, H140K, H140R, H142D, S 148D, S148T, S148N, D152S, Y156H, Y156T, Y156F, E167Y, S168K, R169T, K17 0N, L171K, R172E, R172L, R172W, R172K, R173A, Y175F, Y175W, Q178E, Q178K , T181T, T181A, T181D, T181G, T181N, T181R, T181S, T181E, P188E, P188G, P1 88N, P188S, P188D, P188K, P188R, P188T, G191C, Y205D, Y205N, N265G, E271 K, E271L, N272R, N275E, N278D, N278E, N278G, N278K, F279Y, N280V, H281C, H 281F, H281I, H281L, H281T, H281V, H281M, V283L, Q291K, Q291R, A294D, A29 4L, A294R, A294S, A294T, A294V, A294Y, A294E, A294K, A294N, A294Q, Q298A,Q298S, G301A, G301E, G301H, G301L, G301N, G301Q, G301R, G301S, G301T , G301V, G301Y, G301K, Y302A, Y302G, Y302L, Y302V, Y302W, Y302F, L307L , L307I, N309R, N309A, N309H, N309Q, N309D, N309E, V312L, S314A, K315 E. K315T, K315V, H316A, H316E, H316F, H316L, H316M, H316Q, H316R, H316 V, H316W, H316E, H316Y, L318Q, L318D, L318E, K319L, S320A, T338P, T338Q, T338R, Q340D, Q340K, Q340M, Q340N, Q340R, Q340A, Q340E, Q340P, S356D, S356E, D372P, D372S, S373G, S373L, S373T, Q374D, Q374S, R375D, R375K, R375N, R375T, H406A, H406D, H406P, H406R, and H406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:25. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, the resulting mutants contain the aforementioned substitutions relative to the amino acid sequence shown in SEQ ID NO:26.

[0792] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid alterations include one or more substitutions selected from the group consisting of: A48S, I49A, A52D, A52S, W68N, V101L, A111S, W114E, W114T, T116D, T116E, T116K, T116L, T116M, T116R, T116W, V118Q, A123F, A123L, A123N, A123T, A123Y, D124N, N126C, N126L, R127E, R12 7K, V128A, V128D, V128E, V128I, V128K, V128L, V128P, G131D, G131E, G131K, E132A, E132K, E132P, E132W, H133F, H133Y, R134D, R134E, R134K, R134M, R134N, R134P, R134T, R134W, K136A, K136D, K136E, H140K, H140R, H142D, S148D, S148T, D152S, Y156H, Y156T, E167Y, S16 8K, R169T, K170N, L171K, R172K, R173A, Y175F, Q178E, Q178K, T181E, P188D, P188K, P188R, P188T, G191C, Y205D, N265G, E271K, E271L, N272R, N275E, N278D, N278E, N278G, N278K, F279Y, N280V, H281M, V283L, Q291K, Q291R, A294E, A294K, A294N, A294Q, Q298A, Q298S, G30 1K, Y302F, L307I, N309D, N309E, V312L, S314A, K315E, K315T, K315V, H316Y, L318D, L318E, K319L, S320A, T338P, T338Q, T338R, Q340A, Q340E, Q340P, S356D, S356E, D372P, D372S, S373G, S373L, S373T, Q374D, Q374S, R375D, R375K, R375N, R375T, H406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:25. In some embodiments, for the modification method of an amylase having a similar sequence to Bacillus licheniformis α-amylase or a mutant thereof, the resulting mutant contains the above substitutions relative to the amino acid sequence shown in SEQ ID NO:26.

[0793] In some embodiments, the α-amylase mutant of the present invention may contain amino acid substitutions as described below (1), (2), (3), or (4) or any combination thereof, or may include amino acid substitutions as described below (1), (2), (3), and (4):

[0794] (1) Within the area of ​​region 3, within the area of ​​region 4, and within and around region 6. Amino acid changes within the range, including amino acid substitutions at at least five positions selected from amino acid positions 116-136 (e.g., amino acid substitutions at positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, e.g., amino acid substitutions at positions 5-10, 5-7 or 6-10);

[0795] (2) Amino acid substitutions at at least two positions selected from amino acid positions 175-195 (e.g., amino acid substitutions at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 positions, e.g., amino acid substitutions at 2-4 positions or 3-4 positions);

[0796] (3) Amino acid substitutions at at least 5 positions selected from amino acid positions 296-320 (e.g., amino acid substitutions at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 positions, e.g., amino acid substitutions at 5-8 positions or 6-7 positions);

[0797] (4) Amino acid substitutions at any 1 to 7 positions selected from amino acid positions 271, 274, 281, 294, 340, 356 and 406 (e.g., amino acid substitutions at 1, 2, 3, 4, 5, 6 or 7 positions, e.g., amino acid substitutions at 4-7, 5-7 or 4-6 positions);

[0798] The specific amino acid substitutions at each of the aforementioned positions may be, for example, as described above.

[0799] In some embodiments, the α-amylase mutant of the present invention may contain amino acid substitutions at one or more or all of the following amino acid positions: 116, 123, 126, 128, 132, 156, 181, 265, 278, 281, 301, 309, 316, 318, 320, 340, 356, and 406. In some embodiments, based on any amino acid or amino acid substitution or combination thereof described in this paragraph, the α-amylase mutant of the present invention may further contain amino acid substitutions at one or more or all of the following amino acid positions: 127, 191, 205, and 271. In some embodiments, based on any amino acid or amino acid substitution or combination thereof described in this paragraph, the α-amylase mutant of the present invention may further contain amino acid substitutions at one or more or all of the following amino acid positions: 134, 175, 294, and 302. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further include amino acid substitutions at one or more or all of the following positions: 23, 39, 49, 61, 62, 74, 106, 114, 124, 133, 136, 140, 142, 148, 152, 154, 155, 172, 178, 188, 212, 213, 242, 274, 276, 306, 308, 313, 315, 370, 407, 417, 456. The specific amino acid substitutions at each of these positions may be, for example, as described above.

[0800] In some embodiments, the α-amylase mutant of the present invention may contain amino acid substitutions at one or more or all of the following amino acid positions: 116, 123, 126, 128, 132, 156, 181, 191, 265, 271, 278, 281, 301, 309, 316, 318, 320, 340, 356, and 406. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further contain amino acid substitutions at one or more or all of the following amino acid positions: 127, 175, and 205. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further contain amino acid substitutions at one or more or all of the following amino acid positions: 134, 294, and 302. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further include an amino acid substitution at the following amino acid position: 308. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further include an amino acid substitution at one or more or all of the following amino acid positions: 23, 39, 49, 61, 62, 74, 106, 114, 148, 154, 155, 172, 188, 212, 213, 242, 274, 276, 306, 313, 370, 407, 417, 456. The specific amino acid substitutions at each of these positions may be, for example, as described above.

[0801] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or for mutants thereof, the amino acid alterations may include substitutions at two, three, or four positions selected from amino acid positions 156, 205, 265, and / or 320. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or for mutants thereof, the amino acid alterations do not include combinations of substitutions selected from any of the following:

[0802] H205Y / Y156W;

[0803] H205C / H156Y.

[0804] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid alterations further do not include substitution combinations or amino acid combinations selected from any of the following:

[0805] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 247Y / 360C / 416V / 437W / 126C / 191C / 205D / 31S / 265G;

[0806] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 126C / 191C / 156H / 148N / 265G;

[0807] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 213T / 247Y / 360C / 416V / 437W / 126C / 191C / 156H / 148N / 205D / 31S;

[0808] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 126C / 191C / 320A / 148N / 31S / 265G;

[0809] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 247Y / 360C / 416V / 437W / 126C / 191C / 320A / 148N / 205D;

[0810] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 126C / 191C / 320A / 156H / 31S;

[0811] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 213T / 247Y / 360C / 416V / 437W / 126C / 191C / 320A / 156H / 205D / 265G;

[0812] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 126C / 191C / 265G / 31S;

[0813] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 126C / 191C / 265G;

[0814] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 247Y / 360C / 416V / 437W / 126C / 191C / 148N / 205D / 31S / 265G;

[0815] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 320A;

[0816] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 156H;

[0817] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 265G;

[0818] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 320A / 181Q / 265G / 148N;

[0819] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 320A / 181Q / 49K;

[0820] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 49K / 265G / 148N;

[0821] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 474P / 49K / 320A / 265G;

[0822] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 474P / 181Q / 265G;

[0823] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 474P / 320A / 148N;

[0824] 3(AAPF) / 68W / 114W / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 123N / 142T / 291N / 482P / 134E / 148N / 320A;

[0825] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 49K / 181Q / 148N / 320A.

[0826] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or for mutants thereof, the amino acid alterations do not include substitution combinations selected from any of the following:

[0827] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H247Y / Q360C / D416V / R437W / N126C / G191C / H205D / H31S / N265G;

[0828] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / N126C / G191C / Y156H / S148N / N265G;

[0829] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / K213T / H247Y / Q360C / D416V / R437W / N126C / G191C / Y156H / S148N / H205D / H31S;

[0830] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / N126C / G191C / S320A / S148N / H31S / N265G;

[0831] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H247Y / Q360C / D416V / R437W / N126C / G191C / S320A / S148N / H205D;

[0832] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / N126C / G191C / S320A / Y156H / H31S;

[0833] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / K213T / H247Y / Q360C / D416V / R437W / N126C / G191C / S320A / Y156H / H205D / N265G;

[0834] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / N126C / G191C / N265G / H31S;

[0835] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / N126C / G191C / N265G;

[0836] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H247Y / Q360C / D416V / R437W / N126C / G191C / S148N / H205D / H31S / N265G;

[0837] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / S320A;

[0838] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / Y156H;

[0839] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / N265G;

[0840] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / S320A / T181Q / N265G / S148N;

[0841] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / S320A / T181Q / I49K;

[0842] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / I49K / N265G / S148N;

[0843] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / G474P / I49K / S320A / N265G;

[0844] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / G474P / T181Q / N265G;

[0845] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / G474P / S320A / S148N;

[0846] V3(AAPF) / H68W / D114W / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / A123N / H142T / Q291N / Q482P / L134E / S148N / S320A;

[0847] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / I49K / T181Q / S148N / S320A.

[0848] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, the amino acid alterations may include two, three, or four substitutions selected from the group consisting of: a substitution at amino acid position 156 of 156F, 156H, or 156T; a substitution at amino acid position 205 of 205D or 205N; a substitution at amino acid position 265 of 265G; and / or a substitution at amino acid position 320A of 320A. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, the resulting mutants may include two, three, or four amino acids selected from the group consisting of: 156F, 156H, or 156T; 205D, or 205N; 265G; and 320A. In some embodiments, for the modification of amylases having a sequence similar to that of Bacillus licheniformis α-amylase or mutants thereof, the amino acid alteration may include two, three, or four substitutions selected from the group consisting of: a substitution at amino acid position 156 of Y156F, Y156H, or Y156T; a substitution at amino acid position 205 of Y205D or Y205N; a substitution at amino acid position 265 of N265G; and / or a substitution at amino acid position 320 of S320A.

[0849] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, the amino acid alterations may include two, three, or four substitutions selected from the group consisting of: a substitution at amino acid position 156 of 156H or 156T; a substitution at amino acid position 205 of Y205D; a substitution at amino acid position 265 of 265G; and / or a substitution at amino acid position 320A of 320A. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, the resulting mutants may include two, three, or four amino acids selected from the group consisting of 156H or 156T, 205D, 265G, and 320A. In some embodiments, for the modification of an amylase having a sequence similar to that of Bacillus licheniformis α-amylase or a mutant thereof, the amino acid alteration may include two, three, or four substitutions selected from the group consisting of: a substitution at amino acid position 156 of Y156H or Y156T; a substitution at amino acid position 205 of Y205D; a substitution at amino acid position 265 of N265G; and / or a substitution at amino acid position 320 of S320A.

[0850] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or for mutants thereof, the resulting mutants do not contain one or more amino acids selected from the group consisting of: amino acids at amino acid position 265 being 265A, 265S, 265T, 265V, 265Y, 265C, 265Q, 265E, 265H, 265I, 265L, 265M, 265F, 265P, or 265W; amino acids at amino acid position 156 being 156Y, 156A, 156C, 156Q, 156E, 156G, 156I, 156W, or 156Q; amino acids at amino acid position 156 being 156Y, 156A, 156C, 156Q, 156E, 156G, 156I, 156Q ... 6L, 156M, 156P, 156S, 156T, 156W, or 156V; amino acids at amino acid position 205 are 205C, 205A, 205G, 205I, 205L, 205M, 205F, 205P, 205W, 205Y, or 205V; amino acids at amino acid position 320 are 320R, 320N, 320D, 320C, 320Q, 320E, 320G, 320H, 320I, 320L, 320K, 320M, 320F, 320P, 320S, 320T, 320W, 320Y, or 320V.

[0851] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid alterations do not include one or more substitutions selected from the group consisting of: substitutions at amino acid position 265 of 265A, 265S, 265T, 265V, 265Y, 265C, 265Q, 265E, 265H, 265I, 265L, 265M, 265F, 265P, or 265W; and substitutions at amino acid position 156 of 156Y, 156A, 156C, 156Q, 156E, 156G, 156I, or 156W. L, 156M, 156P, 156S, 156T, 156W, or 156V; substitutions at amino acid position 205 are 205C, 205A, 205G, 205I, 205L, 205M, 205F, 205P, 205W, 205Y, or 205V; substitutions at amino acid position 320 are 320R, 320N, 320D, 320C, 320Q, 320E, 320G, 320H, 320I, 320L, 320K, 320M, 320F, 320P, 320S, 320T, 320W, 320Y, or 320V.

[0852] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid changes may further include substitutions at one, two, three, four, or five positions of amino acid positions 123, 127, 128, 134, and 181; in some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid changes may further include substitutions selected from one or more of the following group: substitutions at amino acid position 123 are: 123E, 123H, 123I, 123K, 123Q, 123E, 123H, 123I, 123K, 123Q, 123E, 123H, 123I, 123Q, 123E, 123I, 123Q ... 3R, 123V, 123F, 123L, 123N, 123T, or 123Y; substitutions at amino acid position 127 are: 127H, 127Q, 127E, or 127K; substitutions at amino acid position 128 are: 128M, 128Q, 128R, 128T, 128A, 128D, 128E, 128I, 128K, 128L, or 128P; The substitutions at amino acid position 134 are: 134A, 134F, 134Q, 134S, 134V, 134D, 134E, 134K, 134M, 134N, 134P, 134T, or 134W; and / or the substitutions at amino acid position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E.

[0853] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, based on any of the amino acid substitutions or combinations thereof described above, the amino acid changes may further include substitutions at one, two, three, four, or five positions of amino acid positions 123, 127, 128, 134, and 181; in some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, based on any of the amino acid substitutions or combinations thereof described above, the amino acid changes may further include substitutions at one, two, three, four, or five positions of amino acid positions 123, 127, 128, 134, and 181. The step includes one or more substitutions selected from the group consisting of: substitutions at amino acid position 123 of 123F, 123L, 123N, 123T or 123Y; substitutions at amino acid position 127 of 127E or 127K; substitutions at amino acid position 128 of 128A, 128D, 128E, 128I, 128K, 128L or 128P; substitutions at amino acid position 134 of 134D, 134E, 134K, 134M, 134N, 134P, 134T or 134W; and / or substitutions at amino acid position 181 of 181E.

[0854] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutants may further include one or more amino acids selected from the group consisting of: amino acids at position 123 being 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123F, 123L, 123N, 123T, or 123Y; and amino acids at position 127 being 127H, 127Q, 127E, or 127... K; amino acids at amino acid position 128 are: 128M, 128Q, 128R, 128T, 128A, 128D, 128E, 128I, 128K, 128L, or 128P; amino acids at amino acid position 134 are: 134A, 134F, 134Q, 134S, 134V, 134D, 134E, 134K, 134M, 134N, 134P, 134T, or 134W; and / or amino acids at amino acid position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E.

[0855] In some embodiments, for the modification method of an amylase having a sequence similar to that of Bacillus licheniformis α-amylase or its mutants, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutant may further include one or more amino acids selected from the group consisting of: amino acid at position 123 being 123F, 123L, 123N, 123T, or 123Y; amino acid at position 127 being 127E or 127K; amino acid at position 128 being 128A, 128D, 128E, 128I, 128K, 128L, or 128P; amino acid at position 134 being 134D, 134E, 134K, 134M, 134N, 134P, 134T, or 134W; and / or amino acid at position 181 being 181E.

[0856] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or for mutants thereof, the amino acid alterations may include substitutions selected from any one of the following groups of amino acids or amino acids selected from any one of the following groups: 156H / 320A; 156H / 205D; 156H / 265G; 205D / 265G; 205D / 320A; 265G / 320A; 156H / 205D / 265G; 156H / 205D / 320A; 205D / 265G / 320A; 156H / 205D / 265G / 320A; 156H / 205D / 265G / 320A / 127K; 156H / 205D / 265G / 320A / 127K / 123N; 156H / 205D / 265G / 320A / 127K / 181E; and 156H / 205D / 265G / 320A / 127K / 123N / 181E.

[0857] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid alterations may include a set of substitutions selected from any one of the following: Y156H / S320A; Y156H / Y205D; Y156H / N265G; Y205D / N265G; Y205D / S320A; N265G / S320A; Y156H / Y205D / N265G; Y156H / Y205D / S320A; Y205D / N265G / S320A; Y156H / Y205D / N265G / S320A; Y156H / Y205D / N265G / S320A / R127K; Y156H / Y205D / N265G / S320A / R127K / A123N; Y156H / Y205D / N265G / S320A / R127K / T181E; and Y156H / Y205D / N265G / S320A / R127K / A123N / T181E.

[0858] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid alteration may further include substitutions at one or more of the following positions: amino acid position 116, 123, 124, 126, 127, 128, 133, 134, 136, 140, 142, 148, 152, 178 and / or 181. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid alteration may further include substitutions at one or more of the following groups: substitution at amino acid position 116 is: 116D, 116K, 116L, 116N, 116Q, 116E or 116M; substitution at amino acid position 123... The substitutions are: 123E, 123H, 123I, 123K, 123Q, 123R, 123T, 123V, 123Y, or 123N; the substitution at amino acid position 124 is: 124N; the substitution at amino acid position 126 is: 126L or 126C; the substitution at amino acid position 127 is: 127H, 127Q, 127K, or 127E; the substitution at amino acid position 128 is: 128I, 128K, 1... 28M, 128Q, 128R, 128T, 128E, 128A, 128D, or 128P; substitution at amino acid position 133 is: 133Y; substitution at amino acid position 134 is: 134A, 134F, 134Q, 134S, 134T, 134V, 134W, 134E, 134N, 134P, 134K, 134D, or 134M; substitution at amino acid position 136 is: 136A; The substitution at amino acid position 140 is 140R; the substitution at amino acid position 142 is 142D; the substitution at amino acid position 148 is 148D or 148N; the substitution at amino acid position 152 is 152S; the substitution at amino acid position 178 is 178K; and / or the substitution at amino acid position 181 is 181T, 181A, 181D, 181G, 181N, 181R, 181S or 181E.

[0859] In some embodiments, for the modification method of an amylase having a sequence similar to that of Bacillus licheniformis α-amylase or a mutant thereof, the amino acid alteration may further include substitutions at one or more of the following positions: amino acid positions 116, 123, 124, 126, 127, 128, 133, 134, 136, 140, 142, 148, 152, 178 and / or 181, based on any of the amino acid substitutions or combinations thereof described above. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid alterations may further include one or more substitutions selected from the group consisting of: a substitution at amino acid position 116 of 116E or 116M; a substitution at amino acid position 123 of 123N; a substitution at amino acid position 124 of 124N; a substitution at amino acid position 126 of 126L; a substitution at amino acid position 127 of 127K or 127E; and a substitution at amino acid position 128 of 127K. 28E, 128A, 128D, or 128P; substitution at amino acid position 133: 133Y; substitution at amino acid position 134: 134E, 134N, 134P, 134K, 134D, or 134M; substitution at amino acid position 136: 136A; substitution at amino acid position 140: 140R; substitution at amino acid position 142: 142D; substitution at amino acid position 148: 148D; substitution at amino acid position 152: 152S; substitution at amino acid position 178: 178K; and / or substitution at amino acid position 181: 181E.

[0860] In some embodiments, for the modification method of amylases having a sequence similar to that of Bacillus licheniformis α-amylase, or mutants thereof, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutant may further include one or more amino acids selected from the group consisting of: amino acid at amino acid position 116 being 116D, 116K, 116L, 116N, 116Q, 116E, or 116M; amino acid at amino acid position 123 being 123E, 123H, 123I, 123K, 123Q, 123R, 123T, 123V, 123Y, or 123N; amino acid at amino acid position 124 being 124N; amino acid at amino acid position 126 being 126L or 126C; amino acid at amino acid position 127 being 127H, 127Q, 127K, or 127E; and amino acid at amino acid position 128 being 128I, 128K, or 128E. 128M, 128Q, 128R, 128T, 128E, 128A, 128D, or 128P; the amino acid at position 133 is 133Y; the amino acid at position 134 is 134A, 134F, 134Q, 134S, 134T, 134V, 134W, 134E, 134N, 134P, 134K, 134D, or 134M; the amino acid at position 136 is 136A; in The amino acid at position 140 is 140R; the amino acid at position 142 is 142D; the amino acid at position 148 is 148D or 148N; the amino acid at position 152 is 152S; the amino acid at position 178 is 178K; and / or the amino acid at position 181 is 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E.

[0861] In some embodiments, for the modification method of amylases having a sequence similar to that of Bacillus licheniformis α-amylase, or mutants thereof, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutant may further comprise one or more amino acids selected from the group consisting of: amino acid 116E or 116M at amino acid position 116; amino acid 123N at amino acid position 123; amino acid 124N at amino acid position 124; amino acid 126L at amino acid position 126; amino acid 127K or 127E at amino acid position 127; and amino acid 128... 128E, 128A, 128D, or 128P; amino acid at position 133 is 133Y; amino acid at position 134 is 134E, 134N, 134P, 134K, 134D, or 134M; amino acid at position 136 is 136A; amino acid at position 140 is 140R; amino acid at position 142 is 142D; amino acid at position 148 is 148D; amino acid at position 152 is 152S; amino acid at position 178 is 178K; and / or amino acid at position 181 is 181E.

[0862] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid alterations may further include substitutions at one or more of the following positions: 132D, 132K, 132T, 132V, 132W, or 132P; 175F or 175W; 188281, 294, 301, 302, 309, 316, 318, 340, 356, and / or 406. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid alterations may further include substitutions at one or more of the following groups: substitution at amino acid position 132: 132D, 132K, 132T, 132V, 132W, or 132P; substitution at amino acid position 175: 175F or 175W; substitution at amino acid position 188... The substitutions are: 188E, 188G, 188N, 188S, 188K, or 188T; the substitutions at amino acid position 281 are: 281C, 281F, 281I, 281L, 281T, 281V, or 281M; the substitutions at amino acid position 294 are: 294D, 294E, 294L, 294R, 294S, 294T, 294V, 294Y, 294N, or 294Q or 294K; the substitutions at amino acid position 301 are: 301A, 301E, ... Substitutions at amino acid position 302 include: 302A, 302G, 302L, 302V, 302W, or 302F; substitutions at amino acid position 309 include: 309R, 309A, 309H, 309Q, 309D, or 309E; substitutions at amino acid position 316 include: 316A, 316E, 316F, 316L, 316M, 301H, 301L, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y, or 301K; substitutions at amino acid position 302 include: 302A, 302G, 302L, 302V, 302W, or 302F; substitutions at amino acid position 309 include: 309R, 309A, 309H, 309Q, 309D, or 309E; substitutions at amino acid position 316 include: 316A, 316E, 316F, 316L, 316M, 301H, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y, or 301K. 16Q, 316R, 316V, 316W, 316E, or 316Y; substitution at amino acid position 318 is 318E, 318Q, or 318D; substitution at amino acid position 340 is 340A, 340D, 340K, 340M, 340N, 340R, 340E, or 340P; substitution at amino acid position 356 is 356D or 356E; and / or substitution at amino acid position 406 is 406A, 406D, 406P, 406R, or 406K.

[0863] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid alterations may further include substitutions at one or more of the following positions: 132, 175, 188, 281, 294, 301, 302, 309, 316, 318, 340, 356, and / or 406. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, in addition to any of the amino acid substitutions or combinations thereof described above, the amino acid alterations may further include substitutions at one or more of the following groups: substitution at amino acid position 132: 132W or 132P; substitution at amino acid position 175: 175F; substitution at amino acid position 188: 188K or 188T; substitution at amino acid position 281: 281M; substitution at amino acid position 282: 132W or 132P; substitution at amino acid position 281: 132W or 132P; substitution at amino acid position 282 ... The substitution at position 294 is 294N, or 294Q, or 294K; the substitution at position 301 is 301K; the substitution at position 302 is 302F; the substitution at position 309 is 309D or 309E; the substitution at position 316 is 316Y; the substitution at position 318 is 318D; the substitution at position 340 is 340E or 340P; the substitution at position 356 is 356D; and / or the substitution at position 406 is 406K.

[0864] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutants may further include one or more amino acids selected from the group consisting of: amino acids at amino acid position 132 being 132D, 132K, 132T, 132V, 132W, or 132P; amino acids at amino acid position 175 being 175F or 175W; amino acids at amino acid position 18... The amino acid at position 8 is 188E, 188G, 188N, 188S, 188K, or 188T; the amino acid at position 281 is 281C, 281F, 281I, 281L, 281T, 281V, or 281M; the amino acid at position 294 is 294D, 294E, 294L, 294R, 294S, 294T, 294V, 294Y, 294N, 294Q, or 294K; and the amino acid at position 301 is 301A, 301E, ... 301H, 301L, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y, or 301K; amino acids at position 302 are 302A, 302G, 302L, 302V, 302W, or 302F; amino acids at position 309 are 309R, 309A, 309H, 309Q, 309D, or 309E; amino acids at position 316 are 316A, 316E, 316F, 316L, 316M, 301Q ... 16Q, 316R, 316V, 316W, 316E, or 316Y; amino acids at amino acid position 318 are 318E, 318Q, or 318D; amino acids at amino acid position 340 are 340A, 340D, 340K, 340M, 340N, 340R, 340E, or 340P; amino acids at amino acid position 356 are 356D or 356E, and / or amino acids at amino acid position 406 are 406A, 406D, 406P, 406R, or 406K.

[0865] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase, or mutants thereof, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutants may further include one or more amino acids selected from the group consisting of: amino acid 132W or 132P at amino acid position 132; amino acid 175F at amino acid position 175; amino acid 188K or 188T at amino acid position 188; and amino acid 28 at amino acid position 281. 1M; amino acids at amino acid position 294 are 294N, 294Q, or 294K; amino acids at amino acid position 301 are 301K; amino acids at amino acid position 302 are 302F; amino acids at amino acid position 309 are 309D or 309E; amino acids at amino acid position 316 are 316Y; amino acids at amino acid position 318 are 318D; amino acids at amino acid position 340 are 340E or 340P; amino acids at amino acid position 356 are 356D, and / or 406K.

[0866] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid changes, in addition to any of the amino acid substitutions or combinations thereof described above, may further include a substitution at amino acid position 278. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid changes, in addition to any of the amino acid substitutions or combinations thereof described above, may further include the following substitutions: 278K, 278D, 278G, or 278E. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid changes, in addition to any of the amino acid substitutions or combinations thereof described above, may further include the following amino acids: 278K, 278D, 278G, or 278E.

[0867] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid changes, in addition to any of the amino acid substitutions or combinations thereof described above, may further include a substitution at amino acid position 278. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid changes, in addition to any of the amino acid substitutions or combinations thereof described above, may further include the following substitutions: 278D, 278G, or 278E. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or their mutants, the amino acid changes, in addition to any of the amino acid substitutions or combinations thereof described above, may further include the following amino acids: 278D, 278G, or 278E.

[0868] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid alterations may further include substitutions at amino acid positions 271 and / or 315, in addition to any of the amino acid substitutions or combinations thereof described above. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid alterations may further include substitutions selected from one or more of the following groups: a substitution at amino acid position 271 of 271K or 271L; and / or a substitution at amino acid position 315 of 315T, 315E, or 315V. In some embodiments, for the modification method of amylase having a sequence similar to that of Bacillus licheniformis α-amylase or its mutants, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutant may further include one or more amino acids selected from the group consisting of: amino acid 271K or 271L at amino acid position 271; and / or amino acid 315T, 315E or 315V at amino acid position 315.

[0869] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid alterations may further include substitutions at amino acid positions 271 and / or 315, in addition to any of the amino acid substitutions or combinations thereof described above. In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid alterations may further include substitutions selected from one or more of the group consisting of: a substitution of 271K at amino acid position 271; and / or a substitution of 315T, 315E, or 315V at amino acid position 315. In some embodiments, for the modification method of amylase having a sequence similar to that of Bacillus licheniformis α-amylase or its mutants, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutant may further include one or more amino acids selected from the group consisting of: amino acid 271K at amino acid position 271; and / or amino acid 315T, 315E or 315V at amino acid position 315.

[0870] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutants may further include the following substitutions relative to the amino acid sequence shown in SEQ ID NO:26:

[0871] A123N / D124N / N126L / H133Y / K136A / H140R / H142D / S148D / D152S / Y156H / Q178K / T181E / Y205D / N265G / S320A.

[0872] In some embodiments, for methods of modifying amylases with sequences similar to Bacillus licheniformis α-amylase or mutants thereof, the amino acid alterations may, based on any of the amino acid substitutions or combinations thereof described above, further include a set of substitutions selected from any of the following, relative to the amino acid sequence shown in SEQ ID NO:26:

[0873] T116E / A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134M / K136A / H140R / H142D / S148D / D152S / Y156H / Q178K / T181E / Y205D / N265G / S320A;

[0874] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0875] T116K / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0876] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0877] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0878] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0879] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0880] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134D / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / E271K / N278D / H281M / A294K / G301K / Y302F / N309D / K315E / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0881] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278G / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0882] T116M / A123N / D124N / N126L / R127K / V128E / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294N / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0883] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0884] T116E / A123N / D124N / N126L / R127K / V128P / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0885] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0886] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294Q / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0887] T116E / A123N / D124N / N126L / R127K / V128A / E132P / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0888] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278E / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0889] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0890] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309E / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0891] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0892] T116E / A123N / D124N / N126L / R127E / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0893] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134D / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0894] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309E / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0895] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0896] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0897] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0898] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0899] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0900] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175...

Claims

1. A method for improving the heat resistance, acid resistance, and / or enzyme activity of α-amylase, comprising altering the following amino acids in the α-amylase: (1) Amino acid changes were performed within region 3; (2) Amino acid changes were made within region 4; (3) Within and / or around Region 6 Amino acid changes within a certain range; or (4) Any combination of (1)-(3) above; in The α-amylase described therein has a substantially identical overall three-dimensional structure to the α-amylase shown in SEQ ID NO:1; Region 3 is the region in the α-amylase that overlaps in three-dimensional structure with amino acids 117-137 of SEQ ID NO:1; Region 4 is the region in the α-amylase that overlaps in three-dimensional structure with amino acids 176-198 of SEQ ID NO:1; Region 6 is the region in the α-amylase that overlaps in three-dimensional structure with amino acids 299-323 of SEQ ID NO:1; Area 6 and surrounding The range refers to the distance between at least one amino acid within the range of region 6 in the three-dimensional structure of the α-amylase and no greater than [amount missing]. Amino acids.

2. The method of claim 1, further comprising altering the amino acid composition of the α-amylase selected from the group consisting of: (1) Amino acid changes within region 3, including the insertion, substitution and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids within region 3; (2) Amino acid changes within region 4, including the insertion, substitution and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids within region 4; (3) In and / or around region 6 Amino acid alterations within the range include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6, and / or around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range; (4) Amino acid changes in region 3 and amino acid changes in region 4, wherein the amino acid changes in region 3 include the insertion, substitution and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids in region 3, and the amino acid changes in region 4 include the insertion, substitution and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids in region 4; (5) Within and / or around Region 6, as well as within and / or around Region 3. Amino acid changes within the specified range, wherein amino acid changes within region 3 include the insertion, substitution, and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 3, and amino acid changes within region 6 include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6, and changes surrounding region 6. Amino acid changes within the range include those around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range; (6) Within and / or around Region 4 and Region 6 Amino acid changes within the specified range, wherein amino acid changes within region 4 include the insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 4, and amino acid changes within region 6 include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6, and changes surrounding region 6. Amino acid changes within the range include those around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range; and (7) Within the area of ​​region 3, within the area of ​​region 4, and around region 6 and / or region 6 Amino acid changes within the specified range, wherein amino acid changes within region 3 include the insertion, substitution, and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 3, and amino acid changes within region 4 include the insertion, substitution, and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids within region 4. Amino acid alterations within region 6 include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6, and are occurring around region 6. Amino acid changes within the range include those around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range.

3. The method of claim 1 or 2, further comprising altering the amino acid composition of the α-amylase selected from the group consisting of: (1) Amino acid changes within region 3, including substitutions of at least 6 amino acids within region 3; (2) Amino acid changes within region 4, including substitution of at least one amino acid within region 4; (3) In and / or around region 6 Amino acid changes within the range, including substitutions of at least three amino acids within region 6 and / or around region 6. Substitution of any 1-7 amino acids within the range; (4) Amino acid changes in region 3 and region 4, wherein the amino acid changes in region 3 include substitutions of at least 7 amino acids in region 3 and the amino acid changes in region 4 include substitutions of at least 2 amino acids in region 4. (5) Amino acid changes in region 3 and region 6, wherein the amino acid changes in region 3 include substitutions of at least 6 amino acids in region 3, and the amino acid changes in region 6 include substitutions of at least 6 amino acids in region 6. (6) Amino acid changes within region 4 and region 6, wherein the amino acid changes within region 4 include the substitution of at least one amino acid within region 4, and the amino acid changes within region 6 include the substitution of at least two amino acids within region 6; and (7) Within the area of ​​region 3, within the area of ​​region 4, and around region 6 and / or region 6 Amino acid changes within the specified range, wherein amino acid changes within region 3 include substitutions of at least 5 amino acids within region 3, amino acid changes within region 4 include substitutions of at least 2 amino acids within region 4, amino acid changes within region 6 include substitutions of at least 5 amino acids within region 6, and changes surrounding region 6... Amino acid changes within the range include those around region 6. The substitution of any 1-7 amino acids within the range.

4. The method according to any one of claims 1-3, wherein the α-amylase has at least 60% sequence identity with SEQ ID NO:1, and Amino acid changes within region 3 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids at positions 117, 118, 119, 120, 121, 122, 124, 125, 127, 128, 129, 130, 132, 133, 134, 135, 136, and 137. Amino acid changes within region 4 include substitutions of any one, two, three, four, five, six, seven, or eight amino acids at positions 176, 177, 178, 179, 184, 188, 191, and 193. Amino acid changes within region 6 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids at positions 300, 302, 304, 305, 307, 309, 310, 311, 312, 313, 316, 317, 318, 319, 321, and 322. Around area 6 Amino acid changes within the range include substitutions of any one, two, three, four, five, six, or seven amino acids at positions 274, 277, 284, 297, 343, 359, and 406. The amino acid position number corresponds to the position number of SEQ ID NO:

1.

5. The method of claim 4, wherein Amino acid substitutions within region 3 include one or more of the following substitutions: The amino acid at position 117 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by W, T, S, M, L, K or E; more preferably by E. The amino acid at position 118 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably V; The amino acid at position 119 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y or Q. The amino acid at position 120 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D or A; The amino acid at position 121 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by M; The amino acid at position 122 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably D; The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y, T, N, or F; more preferably N. The amino acid at position 125 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably N; The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by R or L; The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or E. The amino acid at position 129 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by V, P, L, K, I or A. The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably E or D. The amino acid at position 133 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by W, V, S, P, K, A, or E; and The amino acid at position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably F or E. Amino acid substitutions within region 4 include one or more of the following substitutions: The amino acid at position 177 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L; The amino acid at position 184 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by Q, K or E. The amino acid at position 179 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by S, K, E or A. The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably P; The amino acid at position 191 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with N, K, or D; and The amino acid at position 193 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or F. Amino acid substitutions within region 6 include one or more of the following substitutions: The amino acid at position 304 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably S, N, K or G. The amino acid at position 311 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by T, L, I, F or E. The amino acid at position 312 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, K, E or D. The amino acid at position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably replaced by V, T, L or A; The amino acid at position 318 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by Y, T, L, K, or E. The amino acid at position 319 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by Y, R, K, H, or A. The amino acid at position 321 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E or D; and The amino acid at position 322 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y, K, or H. Around area 6 The amino acid substitutions within the range are selected from one or more of the following substitutions: The amino acid at position 274 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably T, L, K or E. The amino acid at position 277 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L; The amino acid at position 284 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably V or I. The amino acid at position 297 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, K or E. The amino acid at position 343 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by Q, K or E. The amino acid at position 359 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with D; and The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K; The amino acid position number corresponds to the position number of SEQ ID NO:

1.

6. The method according to any one of claims 1-3, wherein the α-amylase has at least 60% sequence identity with SEQ ID NO:26, region 3 includes amino acids at amino acid positions 116-136, region 4 includes amino acids at amino acid positions 175-195, and region 6 includes amino acids at amino acid positions 296-320. Preferably, Amino acid changes within region 3 include substitutions of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acids at positions 116, 118, 123, 124, 126, 127, 128, 131, 132, 133, 134, and 136. Amino acid changes within region 4 include substitutions of any one, two, three, four, or five amino acids at positions 175, 178, 181, 188, and 191. Amino acid changes within region 6 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids at positions 298, 301, 302, 306, 307, 308, 309, 312, 313, 314, 315, 316, 318, 319, and 320; Around area 6 Amino acid changes within the range include substitutions of any one, two, three, four, five, six, or seven amino acids at positions 271, 274, 281, 294, 340, 356, and 406. The amino acid position number corresponds to the position number in SEQ ID NO:

25.

7. The method of claim 6, wherein Amino acid changes within region 3 include substitutions of any one, two, three, four, five, six, seven, eight, nine, or ten amino acids at positions 116, 123, 124, 126, 127, 128, 132, 133, 134, and 136. Amino acid changes within region 4 include substitutions of any one, two, three, four, or five amino acids at positions 175, 178, 181, 188, and 191. Amino acid changes within region 6 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at positions 301, 302, 306, 308, 309, 313, 315, 316, 318, 319, and 320. Around area 6 Amino acid changes within the range include substitutions of any one, two, three, four, five, six, or seven amino acids at positions 271, 274, 281, 294, 340, 356, and 406. The amino acid position number corresponds to the position number in SEQ ID NO:

25.

8. The method of claim 6 or 7, wherein Amino acid substitutions within region 3 include one or more of the following substitutions: The amino acid at position 116 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, Q, E, R, D, M, K, W or L. The amino acid positions 1, 2, and 3 are replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably E, H, I, K, Q, R, V, N, F, Y, L, or T. The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably N; The amino acid at position 126 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L or C. The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by H, Q, E, R, D, M, K, W or L. The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by M, Q, R, T, P, L, K, I, E, D or A. The amino acid at position 131 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D, E or K; The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by D, T, V, W, P, A, or K; and The amino acid at position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, D, E, F, K, W, M, T, E, P, N, D or A; Amino acid substitutions within region 4 include one or more of the following substitutions: The amino acid at position 175 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably F or W. The amino acid at position 178 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or E. The amino acid at position 181 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by T, A, D, E, G, N, R or S. The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by D, E, G, K, N, R, S, or T; and The amino acid at position 191 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably C; Amino acid substitutions within region 6 include one or more of the following substitutions: The amino acid at position 298 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A or S. The amino acid position 301 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, E, H, K, L, N, Q, R, S, T, V or Y. The amino acid at position 302 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, F, G, L, V or W. The amino acid at position 306 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably H, R or Y. The amino acid at position 307 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L or I. The amino acid at position 308 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably replaced by F or M; The amino acid at position 309 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by R, A, D, E, H or Q. The amino acid at position 313 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L; The amino acid at position 314 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A; The amino acid at position 315 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably T or V. The amino acid position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, E, F, L, M, Q, R, V, W, Y or E. The amino acid at position 318 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with Q, E, or D; and The amino acid at position 320 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A; Around area 6 The amino acid substitutions within the range are selected from one or more of the following substitutions: The amino acid at position 271 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or L. The amino acid at position 281 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by C, F, I, L, M, T or V. The amino acid at position 294 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D, E, K, L, N, Q, R, S, T, V or Y. The amino acid at position 340 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, D, E, K, M, N, P or R. The amino acid at position 356 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E or D; and The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably A, D, K, P, or R. The amino acid position number corresponds to the position number in SEQ ID NO:

25.

9. An α-amylase mutant obtained by the method according to any one of claims 1-8.

10. An α-amylase mutant, characterized in that, It includes: (1) Amino acid changes within region 3; (2) Amino acid changes within region 4; (3) Within and / or around Region 6 Amino acid changes within a certain range; or (4) Any combination of (1)-(3) above; in The α-amylase described therein has a substantially identical overall three-dimensional structure to the α-amylase shown in SEQ ID NO:1; Region 3 is the region in the α-amylase that overlaps in three-dimensional structure with amino acids 117-137 of SEQ ID NO:1; Region 4 is the region in the α-amylase that overlaps in three-dimensional structure with amino acids 176-198 of SEQ ID NO:1; Region 6 is the region in the α-amylase that overlaps in three-dimensional structure with amino acids 299-323 of SEQ ID NO:1; Area 6 and surrounding The range refers to the distance between at least one amino acid within the range of region 6 in the three-dimensional structure of the α-amylase and no greater than [amount missing]. amino acids; and The α-amylase mutant exhibits enhanced heat resistance, enhanced acid resistance, and / or enhanced enzyme activity.

11. The α-amylase mutant of claim 10, comprising an amino acid alteration selected from the group consisting of: (1) Amino acid changes within region 3, including the insertion, substitution and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids within region 3; (2) Amino acid changes within region 4, including the insertion, substitution and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids within region 4; (3) In and / or around region 6 Amino acid alterations within the range include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6, and / or around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range; (4) Amino acid changes in region 3 and amino acid changes in region 4, wherein the amino acid changes in region 3 include the insertion, substitution and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids in region 3, and the amino acid changes in region 4 include the insertion, substitution and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids in region 4; (5) Within and / or around Region 6, as well as within and / or around Region 3. Amino acid changes within the specified range, wherein amino acid changes within region 3 include the insertion, substitution, and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 3, and amino acid changes within region 6 include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6, and changes surrounding region 6. Amino acid changes within the range include those around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range; (6) Within and / or around Region 4 and Region 6 Amino acid changes within the specified range, wherein amino acid changes within region 4 include the insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 4, and amino acid changes within region 6 include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6, and changes surrounding region 6. Amino acid changes within the range include those around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range; and (7) Within the area of ​​region 3, within the area of ​​region 4, and around region 6 and / or region 6 Amino acid changes within the specified range, wherein amino acid changes within region 3 include the insertion, substitution, and / or deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids within region 3, and amino acid changes within region 4 include the insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids within region 4. Amino acid alterations within region 6 include the insertion, substitution, and / or deletion of at least 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 amino acids within region 6, and are occurring around region 6. Amino acid changes within the range include those around region 6. The insertion, substitution, and / or deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids within the range.

12. The α-amylase mutant of claim 10 or 11, comprising an amino acid alteration selected from the group consisting of: (1) Amino acid changes within region 3, including substitutions of at least 6 amino acids within region 3; (2) Amino acid changes within region 4, including substitution of at least one amino acid within region 4; (3) In and / or around region 6 Amino acid changes within the range, including substitutions of at least three amino acids within region 6 and / or around region 6. Substitution of any 1-7 amino acids within the range; (4) Amino acid changes in region 3 and region 4, wherein the amino acid changes in region 3 include substitutions of at least 7 amino acids in region 3 and the amino acid changes in region 4 include substitutions of at least 2 amino acids in region 4. (5) Amino acid changes in region 3 and region 6, wherein the amino acid changes in region 3 include substitutions of at least 6 amino acids in region 3, and the amino acid changes in region 6 include substitutions of at least 6 amino acids in region 6. (6) Amino acid changes within region 4 and region 6, wherein the amino acid changes within region 4 include the substitution of at least one amino acid within region 4, and the amino acid changes within region 6 include the substitution of at least two amino acids within region 6; and (7) Within the area of ​​region 3, within the area of ​​region 4, and around region 6 and / or region 6 Amino acid changes within the specified range, wherein amino acid changes within region 3 include substitutions of at least 5 amino acids within region 3, amino acid changes within region 4 include substitutions of at least 2 amino acids within region 4, amino acid changes within region 6 include substitutions of at least 5 amino acids within region 6, and changes surrounding region 6... Amino acid changes within the range include those around region 6. The substitution of any 1-7 amino acids within the range.

13. The α-amylase mutant according to any one of claims 10-12, wherein the α-amylase mutant has at least 60% sequence identity with SEQ ID NO:1, and Amino acid changes within region 3 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids at positions 117, 118, 119, 120, 121, 122, 124, 125, 127, 128, 129, 130, 132, 133, 134, 135, 136, and 137. Amino acid changes within region 4 include substitutions of any one, two, three, four, five, six, seven, or eight amino acids at positions 176, 177, 178, 179, 184, 188, 191, and 193. Amino acid changes within region 6 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids at positions 300, 302, 304, 305, 307, 309, 310, 311, 312, 313, 316, 317, 318, 319, 321, and 322. Around area 6 Amino acid changes within the range include substitutions of any one, two, three, four, five, six, or seven amino acids at positions 274, 277, 284, 297, 343, 359, and 406; and The amino acid position number corresponds to the position number of SEQ ID NO:

1.

14. The α-amylase mutant of claim 13, wherein... Amino acid substitutions within region 3 include one or more of the following substitutions: The amino acid at position 117 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by W, T, S, M, L, K or E; more preferably by E. The amino acid at position 118 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably V; The amino acid at position 119 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y or Q. The amino acid at position 120 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D or A; The amino acid at position 121 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by M; The amino acid at position 122 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably D; The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y, T, N, or F; more preferably N. The amino acid at position 125 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably N; The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by R or L; The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or E. The amino acid at position 129 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by V, P, L, K, I or A. The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably E or D. The amino acid at position 133 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by W, V, S, P, K, A, or E; and The amino acid at position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably F or E. Amino acid substitutions within region 4 include one or more of the following substitutions: The amino acid at position 177 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L; The amino acid at position 184 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by Q, K or E. The amino acid at position 179 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by S, K, E or A. The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably P; The amino acid at position 191 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with N, K, or D; and The amino acid at position 193 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or F. Amino acid substitutions within region 6 include one or more of the following substitutions: The amino acid at position 304 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably S, N, K or G. The amino acid at position 311 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by T, L, I, F or E. The amino acid at position 312 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, K, E or D. The amino acid at position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably replaced by V, T, L or A; The amino acid at position 318 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by Y, T, L, K, or E. The amino acid at position 319 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by Y, R, K, H, or A. The amino acid at position 321 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E or D; and The amino acid at position 322 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y, K, or H. Around area 6 The amino acid substitutions within the range are selected from one or more of the following substitutions: The amino acid at position 274 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably T, L, K or E. The amino acid at position 277 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L; The amino acid at position 284 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably V or I. The amino acid at position 297 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, K or E. The amino acid at position 343 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by Q, K or E. The amino acid at position 359 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with D; and The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K; The amino acid position number corresponds to the position number of SEQ ID NO:

1.

15. The α-amylase mutant according to any one of claims 10-12, wherein the α-amylase mutant has at least 60% sequence identity with SEQ ID NO:26, region 3 includes amino acids at amino acid positions 116-136, region 4 includes amino acids at amino acid positions 175-195, and region 6 includes amino acids at amino acid positions 296-320. Preferably, Amino acid changes within region 3 include substitutions of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acids at positions 116, 118, 123, 124, 126, 127, 128, 131, 132, 133, 134, and 136. Amino acid changes within region 4 include substitutions of any one, two, three, four, or five amino acids at positions 175, 178, 181, 188, and 191. Amino acid changes within region 6 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids at positions 298, 301, 302, 306, 307, 308, 309, 312, 313, 314, 315, 316, 318, 319, and 320; Around area 6 Amino acid changes within the range include substitutions of any one, two, three, four, five, six, or seven amino acids at positions 271, 274, 281, 294, 340, 356, and 406. The amino acid position number corresponds to the position number in SEQ ID NO:

25.

16. The α-amylase mutant of claim 15, wherein... Amino acid changes within region 3 include substitutions of any one, two, three, four, five, six, seven, eight, nine, or ten amino acids at positions 116, 123, 124, 126, 127, 128, 132, 133, 134, and 136. Amino acid changes within region 4 include substitutions of any one, two, three, four, or five amino acids at positions 175, 178, 181, 188, and 191. Amino acid changes within region 6 include substitutions of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at positions 301, 302, 306, 308, 309, 313, 315, 316, 318, 319, and 320. Around area 6 Amino acid changes within the range include substitutions of any one, two, three, four, five, six, or seven amino acids at positions 271, 274, 281, 294, 340, 356, and 406. The amino acid position number corresponds to the position number in SEQ ID NO:

25.

17. The α-amylase mutant of claim 15 or 16, wherein... Amino acid substitutions within region 3 include one or more of the following substitutions: The amino acid at position 116 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by N, Q, E, R, D, M, K, W or L. The amino acid positions 1, 2, and 3 are replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably E, H, I, K, Q, R, V, N, F, Y, L, or T. The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably N; The amino acid at position 126 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L or C. The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by H, Q, E, R, D, M, K, W or L. The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by M, Q, R, T, P, L, K, I, E, D or A. The amino acid at position 131 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D, E or K; The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by D, T, V, W, P, A, or K; and The amino acid at position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, D, E, F, K, W, M, T, E, P, N, D or A; Amino acid substitutions within region 4 include one or more of the following substitutions: The amino acid at position 175 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably F or W. The amino acid at position 178 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or E. The amino acid at position 181 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by T, A, D, E, G, N, R or S. The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by D, E, G, K, N, R, S, or T; and The amino acid at position 191 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably C; Amino acid substitutions within region 6 include one or more of the following substitutions: The amino acid at position 298 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A or S. The amino acid position 301 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, E, H, K, L, N, Q, R, S, T, V or Y. The amino acid at position 302 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, F, G, L, V or W. The amino acid at position 306 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably H, R or Y. The amino acid at position 307 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L or I. The amino acid at position 308 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably replaced by F or M; The amino acid at position 309 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by R, A, D, E, H or Q. The amino acid at position 313 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L; The amino acid at position 314 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A; The amino acid at position 315 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably T or V. The amino acid position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, E, F, L, M, Q, R, V, W, Y or E. The amino acid at position 318 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with Q, E, or D; and The amino acid at position 320 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A; Around area 6 The amino acid substitutions within the range are selected from one or more of the following substitutions: The amino acid at position 271 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably K or L. The amino acid at position 281 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by C, F, I, L, M, T or V. The amino acid at position 294 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by D, E, K, L, N, Q, R, S, T, V or Y. The amino acid at position 340 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably A, D, E, K, M, N, P or R. The amino acid at position 356 is substituted with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E or D; and The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably A, D, K, P, or R. The amino acid position number corresponds to the position number in SEQ ID NO:

25.

18. A polynucleotide encoding an α-amylase mutant according to any one of claims 9-17.

19. A recombinant expression vector comprising the polynucleotide of claim 18.

20. A host cell comprising the polynucleotide of claim 18 or the recombinant expression vector of claim 17.

21. The host cell as described in claim 20, wherein it is a bacterium or a fungus.

22. The host cell of claim 21, wherein the bacteria are derived from the genus Bacillus.

23. The host cell as described in any one of claims 20-22, wherein it is Bacillus subtilis or Bacillus licheniformis.

24. A method for preparing the α-amylase mutant according to any one of claims 9-17, comprising the following steps: (a) Culture the host cells according to any one of claims 18-20 under conditions suitable for the expression of the α-amylase mutant; (b) Recover the expressed α-amylase mutant.

25. An α-amylase mutant obtained by the method of claim 24.

26. A composition comprising the α-amylase mutant of any one of claims 9-17 and one or more other enzymes.

27. The composition of claim 26, wherein one or more additional enzymes are selected from the group consisting of α-amylase, β-amylase, cellulase, glucosylamylase, hemicellulase, isoamylase, isomerase, lipase, phytase, protease, and pullulanase.

28. Use of the α-amylase mutant of any one of claims 9-17 or the composition of any one of claims 24-25 for liquefying starch-containing materials.

29. Use of the α-amylase mutant according to any one of claims 9-17 or the composition according to any one of claims 24-25 for washing.

30. Use of the α-amylase mutant of any one of claims 9-17 or the composition of any one of claims 24-25 for desizing textiles.

31. Use of the α-amylase mutant of any one of claims 9-17 or the composition of any one of claims 24-25 for producing baked goods.

Images