Alpha-amylase mutants, methods for making and using same

Abstract

The present invention provides an α-amylase mutant with improved 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, a method for preparing the same, and its use.

Description

[0001] Cross - reference to related applications This application claims priority to Chinese patent application CN202411997103.X, filed on December 31, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of protein engineering technology, specifically to α-amylase mutants, their preparation methods, and their applications. Background Technology

[0003] Alpha-amylase (EC 3.2.1.1), an important starch-degrading enzyme, catalyzes the α-1,4-glucosidic bonds in starch and its derivatives, producing low-molecular-weight dextrin, maltose, glucose, and other products. Amylase holds a significant position in the global industrial enzyme market, accounting for 30% of the global market share. It is widely used in starch liquefaction processes, food, papermaking, and textile desizing. Despite its broad applications in the starch industry, the susceptibility of α-amylase to high temperatures limits its application scenarios. Gelatinization (100-110℃), liquefaction (80-90℃), and saccharification steps in industrial applications are crucial for the starch processing industry. Therefore, the search for amylases with improved heat resistance and low pH stability is urgent.

[0004] To date, researchers have discovered many α-amylases with commercial value from plants and microorganisms, among which those derived from *Bacillus stearothermophilus* (… 嗜热栖热放线菌 α-Amylases are widely used in the production of amylases due to their excellent heat resistance. However, there is still a need for more novel α-amylases with improved properties, namely α-amylases with higher stability at high temperatures and low pH. Summary of the Invention

[0005] This invention aims to provide an α-amylase mutant with improved thermal stability and / or low pH stability, particularly with enhanced stability under high temperature and low pH conditions compared to parental α-amylase, wild-type α-amylase, or other α-amylase mutants. Based on these improved properties, the α-amylase mutant of this application can produce better results in industrial production, for example, being more suitable for liquefaction reactions in the starch industry and improving liquefaction efficiency. Unless otherwise specified, the amino acid positions described herein correspond to the amino acid positions of SEQ ID NO: 1.

[0006] In one aspect, the present invention provides an α-amylase mutant comprising 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, 273, 274, 276, 277, 278, 281, 282, 284, 293, 294, 296, 297, 300, 302, 304, 305, 307, 309, 310, 311, 312, 313, 316, 317 The mutant has substitutions, insertions, and / or deletions at one or more of the following positions: 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, and 479, wherein the amino acid positions correspond to the positions in SEQ ID NO: 1, wherein the mutant has at least 60% 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, and SEQ ID NO: 9-24, and wherein the mutant has α-amylase activity.

[0007] In some embodiments, the α-amylase mutant of the present invention comprises substitutions at one or more positions of amino acid positions 59, 184, 188, 193, 254, 284, 293, 297 and / or 416. In some embodiments, the substitution at amino acid position 59 is 59A; the substitution at amino acid position 184 is 184E, 184Q or 184K; the substitution at amino acid position 188 is 188P; the substitution at amino acid position 193 is 193K; the substitution at amino acid position 254 is 254S; the substitution at amino acid position 284 is 284V or 284I; the substitution at amino acid position 293 is 293Y; and / or the substitution at amino acid position 416 is 416S.

[0008] In some embodiments, the α-amylase mutant of the present invention comprises substitutions at one or more of the following amino acid positions: 122, 124, 128, 129, 133, 135, 191, 212, and 281. In some embodiments, the substitution at amino acid position 122 is 122D; the substitution at amino acid position 124 is 124N, 124F, 124Y, or 124T; the substitution at amino acid position 128 is 128K or 128E; the substitution at amino acid position 129 is 129I, 129V, 129K, 129A, 129L, or 129P; and the substitution at amino acid position 133 is 133E, 133V, ... 3W, 133P, 133A, 133K or 133S; substitution at amino acid position 135 is: 135E, 135K, 135D, 135T, 135N, 135P, 135W, 135G, 135M or 135R; substitution at amino acid position 191 is: 191D, 191N or 191K; substitution at amino acid position 212 is: 212T; and / or substitution at amino acid position 281 is: 281D.

[0009] In another aspect, the present invention provides a polynucleotide encoding the α-amylase mutant of the present invention, a recombinant expression vector comprising the polynucleotide, and a host cell comprising the α-amylase mutant or the recombinant expression vector. In some embodiments, the host cell is a bacterium. In some embodiments, the bacteria are from the genus Bacillus. In some embodiments, the host cell is Bacillus subtilis or Bacillus licheniformis.

[0010] In another aspect, the present invention provides a method for preparing the above-described α-amylase mutant, comprising the steps of: (a) culturing the host cells described herein under conditions suitable for expression of the α-amylase mutant; and (b) recovering the expressed α-amylase mutant. The present invention also provides an α-amylase mutant obtained by the above method.

[0011] In another aspect, the present invention provides compositions comprising the above-described α-amylase mutant and one or more additional enzymes. In some embodiments, the 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.

[0012] In another aspect, the present invention provides the use of the above-described α-amylase mutant and the above-described composition for liquefying starch-containing materials, for washing, for desizing textiles, and for producing baked products. Detailed Implementation

[0013] 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.

[0014] 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.

[0015] 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%).

[0016] 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."

[0017] 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.

[0018] 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.

[0019] The term "mutant" or "variant" refers to a polypeptide that has one or more amino acid insertions, deletions, and / or substitutions relative to the parent polypeptide. Substitution refers to replacing an amino acid occupying a position with a different amino acid; deletion refers to removing an amino acid occupying a position; insertion refers to adding one or more amino acids (e.g., 1-5) adjacent to an amino acid occupying a position. Mutants or variants retain at least one activity of the parent polypeptide (e.g., α-amino acid). (Amylase activity), but may vary at different activity levels; for example, mutants may exhibit variations in at least one activity or property (e.g., α-amylase activity). The amylase activity remained unchanged or improved relative to the parent polypeptide.

[0020] The term "parent" refers to a polypeptide that undergoes changes 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 derived from any species of microorganism. The terms "...source" or "derived from" mean that the 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. The parent can be a bacterial α-amylase. For example, the parent can be a Gram-positive bacterial polypeptide, such as Bacillus (…). 芽孢杆菌属 Clostridium ( 梭菌属 ), Microbacteria ( 微小杆菌属 ), Enterococcus ( 肠球菌属 ), Bacillus aureus ( 地芽孢杆菌属 ), Bacillus subtilis ( 类芽孢杆菌属 ), Cyclocycline Bacillus ( 嗜酸耐热放线菌属 ), Lactobacillus ( 乳杆菌属 ), Lactococcus ( 乳球菌属 ), Bacillus aureus ( 海洋芽孢杆菌属 ),staphylococcus( 葡萄球菌属 Streptococcus ( 链球菌属 ) or Streptomyces ( 链霉菌属 α-Amylase, or Gram-negative bacterial polypeptides, such as Campylobacter ( 弯曲杆菌属 ), Escherichia coli ( 大肠杆菌 ), Xanthomonas ( 黄杆菌属 ), Clostridium ( 梭杆菌属 ), Helicobacter pylori ( 幽门螺杆菌 ), Colistinia spp. ( 栖热放线菌属 ), Neisseria ( 奈瑟菌属 ), Pseudomonas ( 假单胞菌属 ),salmonella( 沙门氏菌属 ), or ureaplasma ( 脲原体属 α-Amylase. For example, the parent could be *Bacillus stearothermophilus* (…). 嗜热栖热放线菌 ), Bacillus licheniformis ( 地衣芽孢杆菌 Bacillus subtilis ( 枯草芽孢杆菌 ), Bacillus halys ( 嗜盐芽孢杆菌 ), Acetylmicrobacterium ( 乙酰微小杆菌 ), Siberian microbacteria ( 西伯利亚微小杆菌 ), oncolytic spore-forming bacteria ( 解凝胶类芽孢杆菌 ), Bacillus megaterium ( 巨大芽孢杆菌 ), Cyclocarya lactones Sp. 18711 ( 嗜酸耐热放线菌属 种 18711), or Bacillus amyloliquefaciens ( 解淀粉芽孢杆菌 α-Amylase.

[0021] 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. Therefore, the parent α... Amylases can have one or more amino acid substitutions, deletions, and / or insertions. Therefore, the parental α... Amylase can be wild-type α Amylase mutants. The term "parent" as used herein can include, for example, *Bacillus thermophilus* (…). 嗜热栖热放线菌 Wild-type α-amylase (SEQ ID NO: 2), and Bacillus stearothermophilus ( 嗜热栖热放线菌 Mutants of wild-type α-amylase, such as α-amylases having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5. SEQ ID NO: 1 is a mutant of *Bacillus thermophilus* (…). 嗜热栖热放线菌 Wild-type α-amylase (SEQ ID NO:2) lacks its N The terminal 1 to 34 amino acid residues, and the C molecule is missing. Obtained from the terminal 1 to 27 amino acid residues; SEQ ID NO: 5 is based on SEQ ID NO: 1, with the N residue deleted. The 180th and 181st amino acid residues at the end, and their N... The amino acid at the terminal 193 position was mutated to F, and the amino acid at the 416th position was mutated to G. The term "parent" as used herein may include, for example, the α shown in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 9-24. Amylase, 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. Amylase. The term “parent” as used herein may also include polypeptides containing any of the fragments of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 9-24.

[0022] 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%, or 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, and 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).

[0023] 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.

[0024] 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" means that α-amylase or its mutants maintain a certain level of enzyme activity after a period of time at a specific or higher temperature; "low pH stability" means that α-amylase or its mutants maintain a certain level of enzyme activity after a period of time at a lower pH. When comparisons involving stability, for example, "improved stability" means that the percentage of residual enzyme activity of α-amylase or its mutants under specific conditions (e.g., after a period of time at high temperature and / or low pH) is higher than that of other α-amylase mutants and / or parental α-amylases and / or wild-type α-amylases.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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, generating 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., using the Needleman-Wunsch algorithm). For example, using the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276). The Niederman implementation in the Needle program (preferably version 5.0.0 or later) 277 Needleman Wunsch's algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443) 453) 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 (EMBOSS version of BLOSUM62) substitution matrix. Alternatively, the parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.

[0036] "Corresponding to..." 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 optimally aligned with a reference amino acid sequence (e.g., the optimal alignment described above). In this invention, 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: 1. The positions of amino acids in another α-amylase mutant can be determined by using several computer programs that align multiple polypeptide sequences with their respective default parameters. Identification of corresponding amino acid residues in amylases using computer programs including, but not limited to, MUSCLE (multiple sequence comparisons by logarithmic prediction; version 3.5 or later; Edgar, 2004, NucleicAcids 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 using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research, 22:4673) 4680) of EMBOSS EMMA.

[0037] When other α The amylase is dissimilar to the polypeptide in SEQ ID NO:1, rendering traditional sequence-based comparisons ineffective in detecting their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613). 615), other pairwise sequence comparison algorithms can be used. Higher sensitivity in sequence-based searches can be achieved using search procedures that utilize the probabilistic representation (spectrum) of peptide families to search the database. For example, PSI The BLAST program generates multiple spectra through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic AcidsRes. 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) Utilizing multiple sources (PSI) Information such as BLAST, secondary structure prediction, structure alignment spectrum, and solvation potential is used as input to a neural network that predicts the structural folding of a query sequence. Similarly, Gough et al., 2000, J.Mol.Biol.313:903 The 919 method 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 models of proteins have already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms can be used, such as distance alignment matrices (Holm and Sander, 1998, Proteins.33:88). 96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering. 11:739) 747) Compare two or more protein structures, and implementations of these algorithms can also be used to query structure databases containing the target structure to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics. 16:566). 567).

[0038] The mutant representation method of the present invention: amino acids are represented by conventional single-letter or three-letter names. As is known to those skilled in the art, amino acids are named using the following single-letter names: A for alanine (Ala); C for cysteine ​​(Cys); D for aspartic acid (Asp); E for glutamic acid (Glu); F for phenylalanine (Phe); G for glycine (Gly); H for histidine (His); I for isoleucine (Ile); K for lysine (Lys); L for leucine (Leu); M for methionine (Met); N for asparagine (Asn); P for proline (Pro); Q for glutamine (Gln); R for arginine (Arg); S for serine (Ser); T for threonine (Thr); V for valine. The three letters corresponding to these are named Val; W stands for tryptophan, and the three letters corresponding to it are named Trp; and Y stands for tyrosine, and the three letters corresponding to it are named Tyr.

[0039] 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 the 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, you can use " The form "" indicates an amino acid deletion, where 181 means a deletion occurs at position 181 corresponding to the reference sequence, losing the amino acid I that originally occupied that position. The original amino acid preceding the position can be omitted, indicating any amino acid deletion at a specific position, for example, it can be represented by "". The symbol "" indicates the deletion of any amino acid at position 181. In this paper, amino acid substitutions are indicated using the following nomenclature: original amino acid, position, existing amino acid. For example, an amino acid substitution can be represented as "V59A," where 59 indicates that the substitution occurs at the 59th amino acid corresponding to 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. The original amino acid preceding the position can be omitted, indicating that any amino acid at a specific position is substituted with a specific amino acid, or that a specific amino acid is present at a specific position. For example, "59A" can indicate that any amino acid at position 59 is substituted with A, or that amino acid A is present at position 59. Multiple mutations... The changes are connected by " / ", for example, "V59A / N122D / S124N / Q128K" means that amino acid V at position 59 is replaced with A, amino acid N at position 122 is replaced with D, amino acid S at position 124 is replaced with N, and amino acid Q at position 128 is replaced with K. Conversely, "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.

[0040] 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.

[0041] This invention relates to α-amylase mutants possessing α-amylase activity, thermostability, and / or low pH stability, particularly stability at high temperatures and low pH. In some embodiments, the α-amylase mutants of this invention exhibit improved thermostability and / or improved low pH stability compared to parental α-amylase and / or wild-type α-amylase and / or other α-amylase mutants. In some embodiments, the parental α-amylase may be the amino acid sequence shown in SEQ ID NO: 5. 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: 2, SEQ ID NO: 4, or SEQ ID NO: 9-24. In this invention, 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: 1.

[0042] In some embodiments, the α-amylase mutants of the present invention can exhibit significantly improved thermal stability and / or low pH stability compared to parental α-amylase and / or wild-type α-amylase and / or other α-amylase mutants. These properties make them particularly useful in industrial production and can be widely applied in fields such as food, home care, textiles, feed, or medicine, including but not limited to starch liquefaction, saccharification, fermentation, brewing, baking, textile desizing, textile washing, and increased digestibility in animal feed.

[0043] In some embodiments, the α-amylase mutant of the present invention comprises an amino acid alteration 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).

[0044] It should be understood that in this invention, when referring to α-amylase mutants as containing amino acid changes at certain positions, changes at other positions are not excluded. For any position not mentioned, the amino acid at that position may or may not be changed relative to the parental sequence (e.g., SEQ ID NO: 5). In some embodiments, the α-amylase mutant contains only the listed amino acid changes relative to the parental sequence (e.g., SEQ ID NO: 5) and does not contain any amino acid changes at unlisted positions.

[0045] It should be understood that, in this invention, when referring to an α-amylase mutant containing an amino acid change (e.g., amino acid substitution) at a specific position, it can be understood as the mutant containing the described mutated amino acid at that 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 an α-amylase mutant containing an amino acid change (e.g., amino acid substitution) at that position also includes the case where the mutant has no change in the amino acid at that position relative to the corresponding position of the parental α-amylase.

[0046] In some embodiments, the α-amylase mutant of the present invention comprises 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, 273, 274, 276, 277. Substitution, insertion, and / or deletion at one or more positions of SEQ ID NO: 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 positions correspond to SEQ ID NO: 278, 281, 282, 284, 293, 294, 296, 297, 300, 302, 304, 305, 307, 309, 310, 311, 312, 313, 316, 317, 318, 319, 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 The mutant is located at position SEQ ID NO: 1, wherein the mutant has at least 60% 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 wherein the mutant has α-amylase activity.

[0047] 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.

[0048] In some embodiments, the α-amylase mutant of the present invention has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, 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 α-amylase mutant of the present invention is sequence-aligned with the amino acid sequence shown in SEQ ID NO: 5 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide).

[0049] In some embodiments, the α-amylase mutant of the present invention 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, or 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%, or at least 99%, but less than 100%. In some embodiments, the α-amylase mutant of the present invention 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, 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).

[0050] In some embodiments, the α-amylase mutant of the present invention is a mature polypeptide that does not contain a signal peptide and / or a leader sequence. In some embodiments, the α-amylase mutant of the present invention 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.

[0051] In some embodiments, the α-amylase mutant of the present invention comprises substitutions, insertions, and / or deletions at one or more of the following positions: 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; 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. 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. 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; 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; 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; 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. 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. 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; 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. 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; 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; 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; 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. 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; 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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; 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; 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. 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; 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; 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; 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; 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; 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. 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. 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; 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; 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; 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. 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. 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; 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; 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. 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; 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; 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; 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; 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. 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; 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; 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. 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 D; 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. 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. 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. 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. 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; 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. 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; 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; 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. 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. 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; 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. 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; 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. 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. 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; 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; 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. 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. 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. 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; 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; 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; 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; 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; 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; 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. 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. 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; 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; 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; 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. 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; 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; 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; 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; 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. 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; 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; 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; 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. 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; 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; 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; 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; The amino acid position thereon corresponds to the position in SEQ ID NO: 1.

[0052] In some embodiments, the α-amylase mutant of the present invention comprises 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, 129L, 129P, 129V, 130T, 130V, 132D, 132E. 133A, 133E, 133K, 133P, 133S, 133V, 133W, 134E, 134F, 135D, 135E, 135G, 13 5K, 135M, 135N, 135P, 135R, 135T, 135W, 136A, 136V, 137A, 137D, 137K, 138L, 143E, 164F, 164T, 168A, 172E, 176F, 176L, 177L, 178I, 179A, 179E, 179K, 17 9S, 184E, 184K, 184Q, 188P, 191D, 191K, 191N, 193K, 204I, 204V, 206Y, 208N, 212T, 241Y, 254S, 267T, 271K, 273Q, 274E, 274K, 274L, 274T, 276F, 277L, 27 8E, 278K, 281D, 282D, 282N, 282Y, 284I, 284V, 293Y, 294N, 294R, 296A, 297E, 297K, 297N, 300N, 302N, 304G, 304K, 304N, 304S, 305Y, 307L, 309D, 309E, 30 9K, 309N, 310I, 311E, 311F, 311I, 311L, 311T, 312D, 312E, 312K, 312N, 313D, 313G, 316A, 316L, 316T, 316V, 317E, 317R, 317S, 318E, 318K, 318L, 318T, 31 8Y, 319A, 319H, 319K, 319R, 319Y, 321D, 321E, 322H, 322K, 322Y, 333Q, 339E, 341Q, 341R, 343E, 343K, 343Q, 353L, 353Y, 358N, 358S, 359D, 372V, 374A, 37 4K, 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.

[0053] In some embodiments, the α-amylase mutant of the present invention comprises 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, 1 33A, 133E, 133K, 133P, 133S, 133V, 133W, 134E, 134F, 135D, 135E, 135G, 135 K, 135M, 135N, 135P, 135R, 135T, 135W, 136A, 136V, 137A, 137D, 137K, 138L, 1 43E, 164F, 164T, 168A, 172E, 176F, 176L, 177L, 178I, 179A, 179E, 179K, 179 S, 184E, 184K, 184Q, 188P, 191D, 191K, 191N, 193K, 204I, 204V, 206Y, 208N, 2 12T, 241Y, 254S, 267T, 271K, 273Q, 274E, 274K, 274L, 274T, 276F, 277L, 278 E, 278K, 281D, 282D, 282N, 282Y, 284I, 284V, 293Y, 294N, 294R, 296A, 297E, 297K, 297N, 300N, 302N, 304G, 304K, 304N, 304S, 305Y, 307L, 309D, 309E, 30 9K, 309N, 310I, 311E, 311F, 311I, 311L, 311T, 312D, 312E, 312K, 312N, 313D, 313G, 316A, 316L, 316T, 316V, 317E, 317R, 317S, 318E, 318K, 318L, 318T, 31 8Y, 319A, 319H, 319K, 319R, 319Y, 321D, 321E, 322H, 322K, 322Y, 333Q, 339E, 341Q, 341R, 343E, 343K, 343Q, 353L, 353Y, 358N, 358S, 359D, 372V, 374A, 37 4K, 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.

[0054] In some embodiments, the α-amylase mutant of the present invention comprises 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, G132D, G132E, T133A, T133E, T133K, T1 33P, T133S, T133V, T133W, Y134E, Y134F, Q135D, Q135E, Q135G, Q135K, Q135M , Q135N, Q135P, Q135R, Q135T, Q135W, I136A, I136V, Q137A, Q137D, Q137K, A 138L, D143E, V164F, V164T, E168A, L172E, Y176F, Y176L, K177L, F178I, R179 A. R179E, R179K, R179S, A184E, A184K, A184Q, E188P, T191D, T191K, T191N, F193K, L204I, L204V, M206Y, H208N, V212T, F241Y, Q254S, S267T, N271K, L27 3Q, H274E, H274K, H274L, H274T, Y276F, I277L, T278E, T278K, N281D, G282D , G282N, G282Y, M284I, M284V, N293Y, K294N, K294R, Y296A, T297E, T297K, T2 97N, K300N, G302N, A304G, A304K, A304N, A304S, F305Y, M307L, T309D, T309 E. T309K, T309N, L310I, M311E, M311F, M311I, M311L, M311T, T312D, T312E, T 312K, T312N, N313D, N313G, M316A, M316L, M316T, M316V, K317E, K317R, K31 7S, D318E, D318K, D318L, D318T, D318Y, Q319A, Q319H, Q319K, Q319R, Q319Y,T321D, T321E, L322H, L322K, L322Y, E333Q, Q339E, W341Q, W341R, D343E, D343K, D343Q, F353L, F353Y, Q358N, Q358S, E359D, I372V, 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, the α-amylase mutant of the present invention includes the above-described substitutions relative to the amino acid sequence shown in SEQ ID NO: 5.

[0055] In some embodiments, the α-amylase mutant of the present invention may contain substitutions at one or more positions of amino acid positions 59, 184, 188, 193, 254, 284, 293, 297 and / or 416. In some embodiments, the α-amylase mutant of the present invention may contain 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, the α-amylase mutant of the present invention may comprise 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, the α-amylase mutant of the present invention may contain 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. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise 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, the α-amylase mutant of the present invention may further comprise substitutions selected from the group consisting of: a substitution of 122D at amino acid position 122; a substitution of 124N, 124F, 124Y, or 124T at amino acid position 124; a substitution of 128K or 128E at amino acid position 128; and a substitution of 129I, 129V, 129K, 129A, 129L, or 129E at amino acid position 129. 29P; 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, the α-amylase mutant of the present invention 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 129E at amino acid position 129. 29P; Amino acids at amino acid position 133 are: 133E, 133V, 133W, 133P, 133A, 133K, or 133S; Amino acids at amino acid position 135 are: 135E, 135K, 135D, 135T, 135N, 135P, 135W, 135G, 135M, or 135R; Amino acids at amino acid position 191 are: 191D, 191N, or 191K; Amino acids at amino acid position 212 are: 212T; and / or Amino acids 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, the α-amylase mutant of the present invention may contain a group of substitutions or a group of amino acids selected from any of the following: 59A / 188P / 254S / 293Y; 59A / 188P / 254S / 293Y / 212T; 59A / 188P / 254S / 293Y / 281D; and 59A / 188P / 254S / 293Y / 212T / 281D.

[0056] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a group of substitutions selected from any of the following: V59A / E188P / Q254S / N293Y; V59A / E188P / Q254S / N293Y / V212T; V59A / E188P / Q254S / N293Y / N281D; and V59A / E188P / Q254S / N293Y / V212T / N281D.

[0057] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise a substitution at amino acid position 179. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise 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 α-amylase mutant of the present invention may further comprise the following amino acids: 179S, 179E, 179A, or 179K.

[0058] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise 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, the α-amylase mutant of the present invention may further comprise 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, the α-amylase mutant of the present invention may further comprise 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.

[0059] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise substitutions at one or more positions of amino acid positions 304, 311, and / or 319. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise 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 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, the α-amylase mutant of the present invention may further comprise 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.

[0060] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise 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, 267, 271, 273, 274, 276, 277, 278, 282, 2... Substitution at one or more of the following positions: 96, 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, the α-amylase mutant of the present invention may further comprise 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 122: 122D; substitution at amino acid position 124: 124N; substitution at amino acid position 128: 128K; substitution at amino acid position 124: 124N; substitution at amino acid position 128: 128K; substitution at amino acid position 124: 124N; substitution at amino acid position 128: 128K; substitution at amino acid position 124: 124N; substitution at amino acid position 128: 124N; substitution at amino acid position 129 ... The substitutions at position 130 are: 130T or 130V; at position 132, the substitutions are: 132D or 132E; at position 133, the substitutions are: 133V, 133E, 133A, 133K, or 133S; at position 134, the substitutions are: 134E or 134F; at position 135, the substitutions are: 135E, 135K, 135P, or 135D; and at position 137, the substitutions are: 137A, 137... D, 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; Substitution at amino acid position 206: 206Y; Substitution at amino acid position 208: 208N; Substitution at amino acid position 267: 267T; Substitution at amino acid position 271: 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; the substitution at amino acid position 300 is 300N; the substitution at amino acid position 302 is 302N; the substitution at amino acid position 305 is 305Y; and the substitution at amino acid position 307 is 3... 07L; Substitution at amino acid position 309 is: 309D or 309K; Substitution at amino acid position 310 is: 310I; Substitution at amino acid position 312 is: 312D, 312K, 312N or 312E; Substitution at amino acid position 313 is: 313D or 313G; Substitution at amino acid position 316 is: 316T, 316V or 316A; Substitution at amino acid position 317 is: 317R, 317S or 317E; Substitution at amino acid position 318 is: 318Y, 318E, 318T or 318K; Substitution at amino acid position 321 is: 321D; Substitution at amino acid position The substitution at position 322 is 322K; the substitution at position 333 is 333Q; the substitution at position 339 is 339E; the substitution at position 341 is 341R or 341Q; the substitution at position 343 is 343K or 343E; the substitution at position 358 is 358S or 358N; the substitution at position 359 is 359D; the substitution at position 372 is 372V; the substitution at position 374 is 374A or 374K; the substitution at position 375 is 375K or 375F; and the substitution at position 376 is... The substitutions are: 376G; 387I at amino acid position 387; 389T or 389K at amino acid position 389; 392K, 393N or 393Y at amino acid position 392; 403F at amino acid position 403; 406K at amino acid position 406; 426T or 426V at amino acid position 426; 429S at amino acid position 429; 450E at amino acid position 450; 452I at amino acid position 452; and / or 479I at amino acid position 479. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise one or more amino acids selected from the group consisting of: amino acid at position 18 being 18A; amino acid at position 50 being 50S, 50A, or 50E.The amino acid at position 52 is 52Y; the amino acid at position 104 is 104V; the amino acid at position 108 is 108A; the amino acid at position 115 is 115K or 115T; the amino acid at position 117 is 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; and so on. Amino acid at position 132 is 132D or 132E; amino acid at position 133 is 133V, 133E, 133A, 133K, or 133S; amino acid at position 134 is 134E or 134F; amino acid at position 135 is 135E, 135K, 135P, or 135D; amino acid at position 137 is 137A, 137D, or 137K; amino acid at position 143 is 143E; amino acid at position 168 is 168A; amino acid at position 176 is 176F; amino acid at position 204 is 204V or 204I; amino acid at position 206 is 2... 06Y; Amino acid at position 208 is 208N; Amino acid at position 267 is 267T; Amino acid at position 271 is 271K; Amino acid at position 273 is 273Q; Amino acid at position 274 is 274E, 274K, or 274L; Amino acid at position 276 is 276F; Amino acid at position 277 is 277L; Amino acid at position 278 is 278K or 278E; Amino acid at position 282 is 282Y; Amino acid at position 296 is 296A; Amino acid at position 300 is 300N; Amino acid at position 302... The amino acid at position 302N is: 305Y; the amino acid at position 307 is: 307L; the amino acid at position 309 is: 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, 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; and 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 is 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.

[0061] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a set of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO: 5: V59A / A184Q / E188P / Q254S / N293Y; V59A / A184Q / E188P / Q254S / N293Y / N281D / V212T; V59A / A184Q / E188P / Q254S / N293Y / N281D / V212T / E129V / T297N / R179S / T191N / M284V.

[0062] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a set of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO: 5: V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / I372V / Q374A / Y375K; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y / I372V / Q374A / Y375K ; V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y / I372V / Q374A / Y375K; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304G / M311L / T312N / N313G / M316V / K317S / Q319H / I372V / Q374A / Y375K; V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y; 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; 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; 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; 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; 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; 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; V59A / W115T / V119Y / N122D / S124N / Q128K / E129I / G132D / T133V / Q135P / Q137D / E168A / R179K / A184Q / E188P / T191N / F193K / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / W115K / V119Y / N122D / S124N / Q128K / E129I / G132E / T133V / Q135P / Q137D / E168A / R179K / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; V59A / W115K / D117L / V119Y / N122D / S124N / Q128K / E129I / G132D / T133V / Q135E / Q137D / E168A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; V59A / N122D / S124N / Q128K / G132D / T133V / Q135K / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117K / N122D / S124N / Q128K / E129V / G132D / T133E / Q135E / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / N122D / S124N / Q128K / G132E / T133V / Q135K / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117K / N122D / S124N / Q128K / G132E / T133V / Q135E / Q137A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117K / N122D / S124N / Q128K / E129I / G132E / T133V / Q135E / Q137A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / T297E / A304K / F305Y / M311L / T312D / M316A / D318Y / Q319Y / T321D / L322K / S406K; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319H / T321D / S406K; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / V212T / Q254S / S267T / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318T / Q319H / L322K; V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / S267T / N271K / H274E / I277L / T278E / M284I / N293Y / T297N / N313D / D318Y / Q319H / L322K; V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / S267T / H274L / I277L / T278K / M284I / N293Y / T297N / N313D / D318Y / Q319H; V59A / E129V / R179S / A184Q / E188P / V212T / Q254S / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318T / Q319H / L322K; V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / H274K / I277L / T278E / M284I / N293Y / T297N / N313D / D318Y / Q319Y / L322K; V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318Y / Q319H; 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; 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; V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V21 2T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / Q319Y / I372V / Q374A / Y375K; 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; 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; 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; 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; 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; 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; 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; 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; 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; T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204V / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N; T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204I / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N; T50A / V59A / F104V / G108A / E129V / D143E / R179S / A184Q / E188P / T191D / L204V / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N; T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204I / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304N / F305Y / M311L / Q358S / E359D / I389K / S406K / G416S / A426V / Y450E / L452I; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N271K / H274E / T278E / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / D318Y / Q319H; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N271K / H274E / T278K / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / N313D / M316T / D318Y / Q319H; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274L / T278E / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / D318Y / Q319K; 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; D18A / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341R / I372V / Q374K / Y375F; D18A / T50E / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341R / I372V / Q374K; or D18A / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341Q / I372V / Q374A / Y375K.

[0063] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain 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, the α-amylase mutant of the present invention may contain 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, the α-amylase mutant of the present invention may contain one or more amino acids selected from the group consisting of 177L, 212T, and 281D. In some embodiments, the α-amylase mutant of the present invention may contain substitutions selected from the group consisting of D177L, V212T, and N281D.

[0064] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise substitutions at one or more positions of amino acid 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, the α-amylase mutant of the present invention may comprise substitutions selected from one or more of the group consisting of: substitution at amino acid position 117: 117E, 117T, 117W, 117M, 117S, 117L, or 117K; substitution at amino acid position 122: 122D; substitution at amino acid position 124: 124N, 124F, 124Y, or 124T; substitution at amino acid position 128: 128K or 128E; and substitutions at amino acid position 191. The substitutions at 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, the α-amylase mutant of the present invention may 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: 128K or 128E; and amino acid at amino acid position 124 being: 124N, 124F, 124Y, or 124T. The amino acid at position 129 is: 129I, 129K, 129V, 129A, 129L, or 129P; the amino acid at position 133 is: 133E, 133W, 133V, 133P, or 133A; the amino acid at position 135 is: 135E, 135K, 135T, 135D, 135N, 135P, 135W, 135G, 135M, or 135R; the amino acid at position 176 is: 176F or 176L; and / or the amino acid at position 191 is: 191D, 191N, or 191K.

[0065] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention 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 α-amylase mutant of the present invention may further include the following substitution: 274L. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further include the following amino acid: 274L.

[0066] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further include substitutions at one or more of the following positions: amino acid 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, the α-amylase mutant of the present invention may further comprise one or more substitutions selected from the group consisting of: a substitution at amino acid position 118 of 118V; a substitution at amino acid position 119 of 119Q or 119Y; a substitution at amino acid position 120 of 120D or 120A; a substitution at amino acid position 121 of 121M; a substitution at amino acid position 125 of 125N; and a substitution at amino acid position 127 of 128V. 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 164T; at amino acid position 172, the substitution is: 172E; at amino acid position 178, the substitution is: 178I. The substitution at amino acid position 208 is 208N; the substitution at amino acid position 241 is 241Y; the substitution at amino acid position 267 is 267T; the substitution at amino acid position 274 is 274L, 274K, 274E, or 274T; 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, 282N, or 282D; the substitution at amino acid position 294 is 294N or 294R; the substitution at amino... The substitution at position 300 is 300N; the substitution at position 304 is 304K; the substitution at position 309 is 309K, 309N, 309D, or 309E; the substitution at position 311 is 311L, 311E, 311I, 311F, or 311T; the substitution at position 312 is 312D, 312E, or 312K; the substitution at position 313 is 313D or 313G; and the substitution at position 316 is 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; the substitutions at amino acid position 321 are: 321D or 321E; the substitutions at amino acid position 322 are: 322K, 322H or 322Y; the substitutions at amino acid position 343 are: 343E, 343K or 343Q; the substitutions at amino acid position 353 are: 353L or 353Y; the substitution at amino acid position 359 is: 359D; the substitution at amino acid position 403 is: 403F; and / or the substitution at amino acid position 406 is: 406K. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise one or more amino acids selected from the group consisting of: amino acid at position 118 being 118V; amino acid at position 119 being 119Q or 119Y; amino acid at position 120 being 120D or 120A; amino acid at position 121 being 121M; and amino acid at position 125 being... The amino acid at position 125 is 125N; the amino acid at position 127 is 127L or 127R; the amino acid at position 130 is 130V or 130T; the amino acid at position 132 is 132D or 132E; the amino acid at position 134 is 134E or 134F; the amino acid at position 136 is 136V or 136A; the amino acid at position 138 is 138L; and the amino acid at position 164 is 164F or 164. T; The amino acid at position 172 is 172E; the amino acid at position 178 is 178I; the amino acid at position 208 is 208N; the amino acid at position 241 is 241Y; the amino acid at position 267 is 267T; the amino acid at position 274 is 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; the amino acid at position 304 is: 304K; the amino acid at position 309 is: 309K, 309N, 309D, or 309E; the amino acid at position 311 is: 311L, 311E, 311I, 311F, or 311T;The amino acids at position 312 are: 312D, 312E, 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; the amino acids at position 318 are: 318Y, 318E, 318K, 318L, or 318T; and the amino acids at position 319 are: 319R, 319H, 319K, 319E ... 19A or 319Y; amino acid at position 321 is 321D or 321E; amino acid at position 322 is 322K, 322H, or 322Y; amino acid at position 343 is 343E, 343K, or 343Q; amino acid at position 353 is 353L or 353Y; amino acid at position 359 is 359D; amino acid at position 403 is 403F; and / or amino acid at position 406 is 406K.

[0067] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a set of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO: 5: V59A / K177L / E188P / V212T / Q254S / N281D / N293Y; V59A / E129V / K177L / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / K177L / E188P / V212T / Q254S / N281D / N293Y / Y176F / N122D / A184E / Q128K / T191D; and V59A / K177L / E188P / V212T / Q254S / N281D / N293Y / Y176F / N122D / A184E / Q128K / T191D / S124N.

[0068] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a set of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO: 5: V59A / E129I / L172E / Y176F / K177L / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / A118V / E129I / I136A / A138L / V164F / L172E / K177L / F178I / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / A118V / V119Q / D125N / E129I / I136A / A138L / V164F / L172E / Y176L / K177L / F178I / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N; V59A / V119Q / E129I / I136V / V164T / L172E / Y176F / K177L / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N; V59A / D125N / N127L / E129I / I136V / V164F / L172E / K177L / A184Q / E188P / T191D / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N; V59A / D125N / N127L / E129I / I136V / V164F / L172E / K177L / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N; V59A / V119Q / E129I / I136V / V164T / L172E / Y176L / K177L / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127R / Q128K / E129I / I130V / G132E / T133W / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / V119Y / N122D / S124N / D125N / N127L / Q128K / E129I / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274K / T278K / N281D / M284V / N293Y / T297N; 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; V59A / D117E / V119Q / N122D / S124N / Q128K / E129I / G132E / T133W / Q135K / Y176F / K177L / A184E / E188P / T191K / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N; V59A / D117L / N122D / S124N / Q128K / E129I / T133E / Q135W / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274K / T278K / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / D125N / N127L / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / T278E / N281D / M284V / N293Y / T297N; V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / T278K / N281D / M284V / N293Y / T297N; V59A / D117L / N122D / S124N / Q128K / E129I / T133E / Q135W / Y176F / K177L / A18 4E / E188P / T191D / V212T / Q254S / H274K / T278K / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / V212T / Q254S / H274L / I277L / T278K / N281D / M284V / N293Y / T297N; V59A / E129V / Y176F / K177L / A184E / E188P / V212T / Q254S / H274K / I277L / T278K / N281D / M284V / N293Y / T297N / A304K / T309K / M311I / T312D / M316L / K317R / D318E / Q319H; V59A / E129V / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / H274L / T278E / N281D / M284V / N293Y / T297N / A304K / T309N / M311I / T312K / N313D / K317E / D318K / Q319K; V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / T278K / N281D / M284V / N293Y / T297N / A304K / M311T / T312E / K317R / D318E / Q319H; 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; 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; 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; V59A / D117E / N122D / S124N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129L / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128E / E129K / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129P / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124Y / D125N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124T / D125N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124F / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129P / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129P / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135T / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129I / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133V / Q135N / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124F / N127R / Q128K / E129I / T133P / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / D125N / Q128K / E129I / T133E / Q135P / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117T / N122D / S124N / D125N / N127L / Q128E / E129L / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117S / N122D / S124N / Q128K / E129L / T133E / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127R / Q128K / E129V / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129A / I130V / G132D / T133W / Q13 5N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; V59A / D117E / N122D / S124N / N127R / Q128K / E129I / I130V / G132E / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / G132D / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V121M / N122D / S124N / N127L / Q128K / E129A / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129K / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V121M / N122D / S124N / D125N / N127L / Q128K / E129A / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / E120D / N122D / S124N / N127L / Q128K / E129K / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129V / I130V / G132E / T133W / Y134F / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127R / Q128K / E129L / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V119Q / N122D / S124N / D125N / N127L / Q128K / E129P / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129K / G132E / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; V59A / D117T / N122D / S124N / N127R / Q128K / E129I / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117W / V119Q / N122D / S124N / N127L / Q128K / E129I / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / G132E / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / V119Y / N122D / S124N / D125N / N127L / Q128K / E129I / I130T / G132D / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V119Q / N122D / S124N / D125N / N127L / Q128K / E129I / I130V / G132D / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / D125N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V119Y / N122D / S124N / N127L / Q128K / E129I / I130V / G132E / T133A / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117W / V119Y / E120D / N122D / S124N / Q128K / E129I / G132D / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V119Y / N122D / S124N / Q128K / E129I / I130T / G132D / T133A / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124F / N127R / Q128K / E129I / G132D / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117W / V119Y / E120A / N122D / S124N / Q128K / E129I / I130T / G132E / T133E / Y134E / Q135K / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117W / E120D / N122D / S124N / N127L / Q128K / E129L / I130V / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132D / T133W / Q135D / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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 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.

[0069] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise substitutions at one or more of the following positions: amino acid 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, the α-amylase mutant of the present invention may further comprise 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 176: 176F; substitution at amino acid position 135: 135E; substitution at amino acid position 176: 176F; substitution at amino acid position 135: 136E; substitution at amino acid position 176 ... 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, the α-amylase mutant of the present invention 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: 176F; amino acid at position 177: The amino acid at position 177L 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.

[0070] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention 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 α-amylase mutant of the present invention may further include the substitution 316T. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further include the amino acid 316T.

[0071] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise 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, the α-amylase mutant of the present invention may further comprise 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, the α-amylase mutant of the present invention may further comprise amino acids selected from the group consisting of 322K, 343E, or 343K, 359D.

[0072] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise substitutions at one or more positions of amino acid 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, the α-amylase mutant of the present invention may further comprise 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, the α-amylase mutant of the present invention may further comprise one or more amino acids selected from the group consisting of: 282Y, 296A, 300N, 305Y, 309D or 309K, 313D, 317E or 317R, 403F.

[0073] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a set of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO: 5: 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; 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.

[0074] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a set of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO: 5: 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; 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; 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; 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 / ; 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 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.

[0075] In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise substitutions at one or more of the following positions: amino acid 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, the α-amylase mutant of the present invention may further comprise 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: 176F; substitution at amino acid position 177... The substitutions at amino acid position 191 are: 191D; at amino acid position 212, 212T; at amino acid position 274, 274K or 274E; at amino acid position 277, 277L; at amino acid position 281, 281D; at amino acid position 304, 304K; at amino acid position 311, 311L; at amino acid position 312, 312D or 312K; at amino acid position 318, 318Y or 318E; at amino acid position 319, 319H or 319Y; at amino acid position 321, 321D; and 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, the α-amylase mutant of the present invention 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: 176F; amino acid at position 177: ... The amino acid at position 191 is 191D; the amino acid at position 212 is 212T; the amino acid at position 274 is 274K or 274E; 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 or 312K; the amino acid at position 318 is 318Y or 318E; 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.

[0076] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise 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, the α-amylase mutant of the present invention may further comprise 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 acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise amino acids selected from the group consisting of: an amino acid at amino acid position 316 of 316T; an amino acid at amino acid position 343 of 343 of 343E or 343K; and / or an amino acid at amino acid position 359 of 359D.

[0077] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise a substitution at amino acid positions 296 and / or 322. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise one or more substitutions selected from the group consisting of 296A and 322K. In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise one or more amino acids selected from the group consisting of 296A and 322K.

[0078] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise substitutions at one or more positions of 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, the α-amylase mutant of the present invention may further comprise 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, the α-amylase mutant of the present invention may further comprise 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.

[0079] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a set of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO: 5: 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; 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.

[0080] In some embodiments, based on any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may contain a set of substitutions selected from any of the following relative to the amino acid sequence shown in SEQ ID NO: 5: 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; 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; 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; 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 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.

[0081] Based on any of the amino acids or amino acid substitutions or combinations thereof described above, any parental or α-amylase mutant of the present invention may further include one or more amino acid changes selected from the group consisting of: , 193F and 416G. In some embodiments, any parent or α-amylase mutant of the present invention may further comprise the following amino acid alterations: In some embodiments, any parental or α-amylase mutant of the present invention may further comprise the following amino acid alterations: .

[0082] The α-amylase mutant of the present invention possesses α-amylase activity and stability at high temperatures and / or low pH. In some embodiments, the α-amylase mutant of the present invention exhibits improved stability at high temperatures and / or low pH compared to parental α-amylase, wild-type α-amylase, or other α-amylase mutants. "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, more preferably 95°C-110°C. "Low pH" refers to a pH value of 4.0-5.8, for example, 4.0-5.0, preferably 4.0-4.5, more preferably 4.0-4.2.

[0083] 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% higher. In some embodiments, the parental α-amylase or other α-amylase mutant may be any of the α-amylases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 9-24. Amylase. 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.

[0084] In some embodiments, the α-amylase mutant of the present invention exhibits higher residual enzyme activity or tolerance factor at high temperatures, particularly above 80°C (e.g., 80°C-115°C, 85°C-110°C, or about 100°C) than the parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same conditions. In some embodiments, the α-amylase mutant of the present invention exhibits higher residual enzyme activity or tolerance factor at 100°C than the parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same conditions. In some embodiments, the α-amylase mutant of the present invention exhibits higher residual enzyme activity or tolerance factor after being held at 100°C for 30 minutes than the parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same conditions. In some embodiments, the parental α-amylase or other α-amylase mutant is any one of the α-amylases described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 9-24. Amylase. In some embodiments, the enzyme activity of untreated mutants is measured at 70°C.

[0085] 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.

[0086] 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.

[0087] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention under low pH conditions, particularly pH 4.0-5.8 (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 % 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 % of the α-amylase mutant of the present invention after being kept 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 parental α-amylase or other α-amylase mutant is any one of the α-amylases described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 9-24. Amylase. In some embodiments, the enzyme activity of untreated mutants is measured at pH 6.0.

[0088] 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.

[0089] 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.

[0090] 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 kept 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 embodiments, the parental α-amylase or other α-amylase mutant is any one of the α-amylases described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 9-24. Amylase. In some embodiments, the enzyme activity of untreated mutants is measured at 70°C and pH 6.0.

[0091] 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.

[0092] 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.

[0093] The present invention also relates to methods for improving thermal stability and / or low pH stability, including the use of parental α-amylases (e.g., but not limited to, the α-amylases described in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 9-24). The above mutations can be performed using amylase. Any method known in the art can be used to perform these mutations, such as site-directed mutagenesis, homologous recombination, etc.

[0094] The present invention also relates to a polynucleotide encoding the α-amylase mutant of the present invention, and a recombinant expression vector comprising the polynucleotide for expressing the α-amylase mutant. The polynucleotide may further comprise a sequence encoding a signal peptide to allow the mutant to be secreted extracellularly after expression.

[0095] The expression vector comprises a polynucleotide encoding an α-amylase mutant of the present invention, operatively linked to one or more control sequences that direct the expression of the coding sequence of the α-amylase mutant in a suitable host cell. Control sequences include, but are not limited to, promoters, terminators, leader sequences, polyadenylated sequences, signal peptide sequences, etc. These control sequences may be natural or synthetic. Constitutive or inducible promoters may be used. Control sequences suitable for different host cells are well known to those skilled in the art. In some embodiments, the expression vector of the present invention comprises a natural or synthetic promoter sequence, a natural or synthetic ribosome binding site, and a natural or synthetic terminator sequence, these genetic elements together with a synthetic α-amylase mutant coding sequence to form an expression component, which, together with the vector backbone, constitutes the expression vector. For example, the expression vector includes an expression component comprising: a promoter sequence, a synthetic ribosome binding site, a synthetic nucleotide sequence encoding the α-amylase mutant of the present invention, and a terminator sequence. The signal peptide sequence can guide the secretion of α-amylase mutants. Introducing the signal peptide sequence into an expression vector or expression component, especially upstream of the start codon, is more conducive to the secretion of α-amylase mutants. In some embodiments, the promoter may be the nucleotide sequence shown in SEQ ID NO: 6. In some embodiments, the terminator may be the nucleotide sequence shown in SEQ ID NO: 7. In some embodiments, the signal peptide sequence may be the nucleotide sequence shown in SEQ ID NO: 8.

[0096] The expression vector can be a self-replicating vector, i.e., one that replicates independently of the chromosome, such as a plasmid or artificial chromosome, or it can be a vector that integrates into the genome and replicates along with the chromosome when introduced into a host cell. The expression vector may contain one or more selectable markers that allow convenient selection of transformed, transfected, or transduced cells; these markers may provide, for example, resistance to antibiotics or heavy metals. The expression vector may also contain an origin of replication to allow the expression vector to replicate autonomously within the host cell. The selection of these elements is a common technique in the art, and the methods for constructing the recombinant expression vector of the present invention using these elements are well known in the art, for example, see J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition. In some embodiments, the expression vector is the pYF-tsDE plasmid disclosed in Example 1 of Chinese Patent Publication No. CN104073458A.

[0097] This invention also relates to recombinant host cells for expressing the α-amylase mutant of the invention. The host cell can be a prokaryotic or eukaryotic cell, such as a bacterial cell, fungal cell, plant cell, insect cell, or animal cell (e.g., mammalian cell). In some embodiments, the host cell can be a fungus, such as one from the genus *Aspergillus*. 曲霉属 Fungi, such as Aspergillus niger ( 黑曲霉 Aspergillus oryzae ( ) 米曲霉 In some embodiments, the host cell may be bacteria, preferably from the genus Bacillus (Bacillus). 芽孢杆菌属 Bacteria, such as Bacillus subtilis ( 枯草芽孢杆菌 ), Bacillus licheniformis ( 芽孢杆菌属 地衣状 ), Bacillus megaterium ( 巨大芽孢杆菌 Genetically engineered strains used as host cells for recombinase products have been reported in the literature (Widner et al., Journal of Industrial Microbiology & Biotechnology, 25, 204). 212, 2000). In a preferred embodiment, using 地衣芽孢杆菌 As host cells. Methods for introducing polynucleotides or expression vectors into host cells are well known to those skilled in the art, such as through competent cell transformation, electroporation, and protoplast transformation (Young et al., J Bacteriology, 81, 823). 829, 1961; Shigekawa et al., Biotechniques, 6, 742 751, 1988; Chang et al., Molecular General Genetics, 168, 111 115, 1979), etc. In a preferred embodiment, the expression vector can be integrated into Bacillus licheniformis ( ). 地衣芽孢杆菌 On the genome of ).

[0098] The recombinant host cells of this invention are capable of producing α-amylase mutants. In some embodiments, the recombinant host cells contain at least one copy of a nucleotide sequence encoding an α-amylase mutant, which is capable of expressing the α-amylase mutant under suitable conditions. In some embodiments, the recombinant host cells can be genetically engineered to contain one or more nucleotide sequences encoding the α-amylase mutant gene expression. Any technique can be used to genetically engineer host cells to contain one or more nucleotide sequences capable of encoding the α-amylase mutant of this invention, for example, through chromosomal integration. In some embodiments, vectors containing temperature-sensitive origin and resistance selection markers can be used in the integration step. In some embodiments, these vectors are integrated into specific regions of the genome via the Campbell's mechanism, and recombinant bacteria are obtained through resistance selection, with the resistance selection markers being removed by homologous recombination during subsequent culture.

[0099] In some embodiments, the recombinant host cell is genetically engineered to inactivate one or more endogenous proteins. Endogenous proteins that can be inactivated include, but are not limited to, extracellular proteases. In some embodiments, the recombinant host cell inactivates one or more endogenous proteins before or after transformation to contain the nucleotide sequence of an α-amylase mutant expression gene. In a preferred embodiment, exogenous secreted proteases of the host cell (e.g., bacteria) are inactivated before the vector containing the α-amylase mutant expression gene is introduced. In some embodiments, the host cell is *Bacillus licheniformis* (…). 地衣芽孢杆菌 Furthermore, some exogenous protease genes can be inactivated through modification. In a preferred embodiment, the host cell is Bacillus licheniformis (…). 地衣芽孢杆菌 It can also inactivate one or more extracellular proteases, such as, but not limited to, subtilisin (AprE) and glutamate-specific proteases like glutamic acid. Specific protease (Blase). These genetic engineering modifications make Bacillus licheniformis (… B. 地衣状 The strain is more suitable for the expression and secretion of α-amylase mutants.

[0100] The present invention also relates to a method for preparing the above-described α-amylase mutant, comprising: (a) culturing host cells of the present invention under conditions suitable for expression of the α-amylase mutant; and (b) recovering the α-amylase mutant. In some embodiments, the expression of the α-amylase mutant in the host cells may be constitutive or inducible, depending on the control sequence (e.g., a promoter) used to direct the expression of the α-amylase mutant. When the expression of the α-amylase mutant in the host cells is inducible, step (a) may further include inducing the expression of the α-amylase mutant, the inducer used depending on the control sequence (e.g., a promoter) used to direct the expression of the α-amylase mutant.

[0101] In some embodiments, the method for preparing the α-amylase mutant includes: (a) inserting a polynucleotide encoding the α-amylase mutant of the present invention into a plasmid vector to prepare a recombinant expression vector for expressing the α-amylase mutant; (b) transforming the recombinant expression vector prepared in step (a) into Bacillus licheniformis (B. licheniformis). 地衣芽孢杆菌 (c) Obtain host cells expressing the α-amylase mutant in a culture; (d) Culture the host cells to express the α-amylase mutant; (e) Recover the α-amylase mutant. In some embodiments, the host cells are *Bacillus licheniformis* (…). 地衣芽孢杆菌 The resistance selection gene is removed, making it environmentally harmless and producing α-amylase mutants that are more suitable for use in the food industry.

[0102] Suitable conditions for expressing the α-amylase mutant are well known to those skilled in the art and can be selected according to the type of host cell. For example, host cells can be cultured using a nutrient medium containing a carbon source, nitrogen source, and / or inorganic salts, and growth conditions such as temperature and / or nutrient supply can be controlled. In some embodiments, host cells are cultured at 37°C. Host cells can be cultured in shake flasks or in laboratory or industrial fermenters, such as continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation. In some embodiments, host cells are cultured by shake flask fermentation, preferably at 220 rpm.

[0103] The α-amylase mutant can be secreted into the culture medium and subsequently recovered from it. The α-amylase mutant can be recovered using methods known in the art. For example, the α-amylase mutant can be recovered from the nutrient medium using a variety of conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, and / or precipitation. The α-amylase mutant may also not be secreted, i.e., expressed and maintained intracellularly; in this case, the α-amylase mutant can be recovered after cell lysis. In some embodiments, the α-amylase mutant is recovered by collecting the fermentation broth.

[0104] The enzyme activity of the generated α-amylase (including the α-amylase mutant described herein) can be detected using a variety of methods known in the art. For example, the enzyme activity of α-amylase can be determined by detecting its activity in catalyzing starch hydrolysis. The enzyme activity assay can be, for example, an iodine-starch colorimetric method, in which 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, α-amylase activity is detected at 70°C and pH 6.0.

[0105] The α-amylase mutant of this invention exhibits high enzyme activity, good thermal stability, and / or stability under low pH conditions, and can be widely used in various fields, such as food, home care, textiles, animal feed, or pharmaceuticals. The application of α-amylase in these fields is known, and the α-amylase of this invention, due to its superior performance, will achieve better results than parental α-amylase, wild-type α-amylase, or other α-amylase mutants in these applications.

[0106] Those skilled in the art will understand that in industrial production, starch liquefaction is typically carried out at high temperatures (e.g., above 80°C, such as 80°C-115°C) and low pH (e.g., pH 4.0-5.0). Therefore, the α-amylase provided in this application, which exhibits higher stability at high temperatures and low pH, will be particularly useful in liquefying starch-containing materials. It can maintain high enzyme activity (i.e., the activity of hydrolyzing α-1,4-glycosidic bonds in starch molecules to generate dextrins, oligosaccharides, and monosaccharides) at high temperatures and / or low pH, thereby improving starch liquefaction efficiency, optimizing starch liquefaction effects, and reducing the amount of enzyme added while achieving comparable results to other α-amylases.

[0107] In fields including but not limited to those described above (e.g., starch liquefaction), the α-amylase mutants of the present invention can be used in combination with other enzymes, and the composition may contain one or more of the α-amylase mutants of the present invention, and one or more other enzymes. In some embodiments, the other enzymes are one or more selected from the group consisting of: α-amylase, β-amylase, cellulase, glucosylamylase, hemicellulase, isoamylase, isomerase, lipase, phytase, protease, and pullulanase. In some embodiments, the α-amylase among the other enzymes differs from the α-amylase mutants of the present invention contained in the composition, for example, by having different sequences, different sources, and / or different functional activities.

[0108] In some embodiments, the α-amylase mutants or compositions of the present invention can be used to liquefy starch or starch-containing materials, and thus can be used in washing, textile desizing, and the production of baked goods. For example, in the food industry, α-amylase can be used in starch processing, alcohol brewing, and bread baking, specifically for starch liquefaction, saccharification, production of high-maltose syrup, and production of high-glucose syrup. For example, in the home care field, α-amylase can be used to remove starch-containing dirt and stains in dishwashing and laundry methods. For example, in the textile field, α-amylase can be used to degrade starch slurries, improving desizing efficiency. For example, in the feed field, α-amylase can be used as a feed additive to increase digestibility and improve the nutritional value of feed. For example, in the pharmaceutical field, α-amylase can be used to prepare drug carriers, sustained-release agents, etc.

[0109] The present invention is further described through the following embodiments, which should not be construed as limiting the invention. Unless otherwise specified, all reagents used in the following embodiments are commercially available products.

[0110] Example 1: Construction of pYF-tsDE plasmid The construction process of the pYF-tsDE plasmid can be found in Example 1 of Chinese Patent Publication No. CN104073458A.

[0111] Example 2: Protease-deficient Bacillus licheniformis ( 地衣芽孢杆菌 Construction of strains Genetically engineered strains used as host cells for recombinase products have been reported in the literature (Widner et al., Journal of Industrial Microbiology & Biotechnology, 25, 204). 212, 2000). These recombinant host cells generally contain one or more nucleic acid structures encoding the target sequence for enzyme expression. In this invention, Bacillus licheniformis ( 地衣芽孢杆菌Bacillus ( ) is used as a recipient bacterium for gene manipulation. 芽孢杆菌属 Transformation of cells can currently be achieved through very mature methods, such as competent cell transformation, electroporation, and protoplast transformation (Young et al., J Bacteriology, 81, 823). 829, 1961; Shigekawa et al., Biotechniques, 6, 742 751, 1988; Chang et al., Molecular General Genetics, 168, 111 115, 1979).

[0112] In this invention, a single α-amylase mutant expression cassette includes a natural or synthetic promoter sequence, a signal peptide sequence screened from bacilli, a synthetic ribosome binding site, and a signal peptide sequence derived from *Bacillus stearothermophilus*. 嗜热栖热放线菌 The design replaces the α-amylase mutant encoding gene of *Bacillus licheniformis* with a transcription terminator. This design will significantly enhance gene expression levels and the secretion of the α-amylase mutant in the host strain. 地衣芽孢杆菌 Specific sites on the cell genome are achieved through plasmid-mediated single crossover homologous recombination.

[0113] In Bacillus licheniformis ( 地衣芽孢杆菌 In this context, the activity of extracellular proteases is detrimental to the secretion of heterologous enzymes. Two main extracellular proteases have been identified: subtilisin (AprE) and glutamic acid-specific proteases. Specific protease, Blase), in Bacillus licheniformis ( 地衣芽孢杆菌 Most of the extracellular protease activity in ) originates from these two proteases.

[0114] In this invention, to obtain the structural integrity of α-amylase mutant gene expression, the two genes mentioned above are inactivated using a continuous single-crossover Campbellian mechanism. The construction process can be referred to Example 2 of Chinese Patent Publication No. CN107345224A. The specific operations are as follows: 2.1 pYF tsDE was digested with BglII and then treated with heat-sensitive calf intestinal alkaline phosphatase (CIP) to inhibit self-ligation; 2.2 Gene knockout (1) In order to obtain each gene deletion fragment, approximately 500 bp homologous sequences were amplified from both sides of the gene to be knocked out using PCR with Bacillus licheniformis genomic DNA (CICC22794, purchased from the China Microbial Culture Collection) as a template. Single clones of Bacillus licheniformis were pre-denatured at 98°C for 5 minutes and then used directly as genomic DNA templates in the PCR reaction.

[0115] The primers used for the PCR reaction were synthesized by Genscript. The primer sequences are as follows: The primers for amplifying the upstream sequence of the AprE gene are: AprE-CZ-F1TTATTGAGCGGCAGCTTCGACATTGATCAGACCTT (SEQ ID NO: 25) AprE_R1CCTTACGGCATTCCTCTCAACAGCGGATCTTCAG (SEQ ID NO: 26) The primers for amplifying the downstream sequence of the Apr gene are: AprE_F2CCTGAAGATCCGCTGTTGAGAGGAATGCCGTAAGG (SEQ ID NO: 27) AprE-CZ-R2ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGAC (SEQ ID NO: 28) The primers for amplifying the upstream sequence of the Blase gene are: Blase-CZ-F1TTATTGTGCGCTGTTTTTCCAGTTGGTCAAATTGTCG (SEQ ID NO: 29) Blase_R1CGGACAAGGGTCACCAACGGGACAACTGTTACCATC (SEQ ID NO: 30) The primers for amplifying the downstream sequence of the Blase gene are: Blase_F2GATGGTAACAGTTGTCCCGTTGGTGACCCTTGTCC (SEQ ID NO: 31) Blase-CZ-R2CGGCGTTGGTTAGTAAAAAGAGTGTTAAACGAGGTTTGAT (SEQ ID NO: 32) The PCR amplification system was 50 μL, and the reaction procedure was as follows: (1) Pre-denaturation of Bacillus licheniformis CICC22794 monoclonal strain at 98°C for 8 minutes; (2) 96℃, 15 seconds; (3) 58℃, 15 seconds; (4) 72℃, 30 seconds; repeat steps 2-4 25-30 times; (5) Extend to 72°C for 2 minutes.

[0116] The PCR products were detected by 0.8% agarose gel electrophoresis and then purified using the Zymoclean™ Gel DNA Recovery Kit (ZYMO RESEARCH, Cat. No. D4002).

[0117] 2.3 Overlap PCR method for amplifying the target gene with an internal sequence deletion of approximately 400-500 bp. The deleted gene fragments were obtained using overlap extension PCR (SOE), and the specific procedure is as follows: (1) The upstream and downstream PCR fragments of each gene in section 2.2 were recovered and purified respectively; (2) Using a 1:1 molar ratio of upstream and downstream homologous sequence fragments of each target gene as a template, the AprE gene or Blase gene with internal deletion fragments was amplified by PCR using primers XX-CZ-F1 and XX-CZ-R2 ("XX" represents AprE or Blase).

[0118] The above fragments were then recombined into the BglII-linearized pYF-tsDE vector using the GenBuilder™ Plus Cloning Kit (Genscript, catalog number: L00744). The resulting recombinant plasmids were named pYF-tsDE-AprE and pYF-tsDE-Blase, respectively. These recombinant plasmids are temperature-sensitive, and the AprE or Blase genes contained within them have a deletion of approximately 400-500 bp compared to the complete gene.

[0119] 2.4 Plasmid Transformation The method and screening process for transforming the knockout plasmid into Bacillus licheniformis competent cells used in this experiment are as follows: (1) Transform Bacillus licheniformis CICC22794 competent cells with thermosensitive plasmids pYF-tsDE-AprE or pYF-tsDE-Blase; (2) Positive clone strains were screened for erythromycin (5 μg / mL) resistance on LB medium (containing 10 g peptone, 5 g yeast extract and 10 g sodium chloride per liter) at 30 °C. (3) The positive clone strain is then transferred to a 37°C environment for culture, so that the temperature-sensitive plasmid can fuse into the host genome. In order to make the gene replace at the set site, several clones are selected and inoculated into 2×YT medium and cultured continuously for 24 hours, and then subcultured again. The whole process is subcultured 4-5 times (generally requiring 5-7 days).

[0120] (4) Screen for erythromycin-sensitive Bacillus licheniformis cells for PCR identification. At the same time, observe the clear hydrolysis zone on 1% skim milk LB plates. The knockout strain should show a significantly reduced hydrolysis zone.

[0121] PCR primers used for identification: AprE: AprE-seqF1 / AprE-seqR3 Blase: Blase-seqF1 / Blase-seqR3 AprE-seqF1:GCCAGGTTGAAGCGGTCTATTCAT (SEQ ID NO: 33) AprE-seqR3:TACGGCCATCCGACCATAATGGAAC (SEQ ID NO: 34) Blase-seqF1:GAAGAGCCGGTCACAATTGC (SEQ ID NO: 35) Blase-seqR3: GGCCGTTAGATGTGACAGCC (SEQ ID NO: 36) Example 3: Integration and Construction of α-Amylase Mutant Strains 3.1 Construction of parental α-amylase expression cassette The construction process was based on Example 3 of Chinese Patent Publication No. CN107345224A, and is as follows: The amino acid sequence of the parental α-amylase is shown in SEQ ID NO: 5, the promoter sequence is SEQ ID NO: 6, the synthesized ribosome binding site is aaaggagg, and the synthesized termination sequence is SEQ ID NO: 7. A strong natural signal peptide sequence (SEQ ID NO: 8) screened from Bacillus subtilis was inserted upstream of the promoter of the parental α-amylase (SEQ ID NO: 5) encoding gene to enhance the secretion efficiency of the expressed enzyme. The complete α-amylase expression cassette was inserted into the BglII site of the linearized pYF-tsDE using the GenBuilder™ Plus Cloning Kit (provided by Genscript, catalog number: L00744). The resulting thermosensitive integrative plasmid was named pYF-tsINT-AmyE. The synthesis of the above sequences was performed by Genscript, and the α-amylase expression cassette was obtained by seamlessly cascading the above sequences. 3.2 Plasmid transformation and strain construction The above-mentioned integrated plasmid pYF-tsINT-AmyE was transformed into Bacillus licheniformis (CICC22794, purchased from China Microbial Culture Collection) lacking the AprE and Blase protease genes. The α-amylase expression cassette without the resistance marker will replace the original AmyE expression cassette. Using the above method, a strain that successfully integrated the α-amylase encoding gene into the Bacillus licheniformis chromosome produced a clear zone on blue starch agar plates (composed of starch hydrolysis medium and iodine solution; starch hydrolysis medium: 10g soluble starch, 1g MgCO3, 0.3g K2HPO4, 1g KNO3, 0.5g NaCl, 20g agar, 1000ml distilled water, pH 7.0; iodine solution preparation: 1g iodine flakes, 2g potassium iodide, 1000ml distilled water, pH 7.2–7.4; the test strain was inoculated onto starch hydrolysis agar plates using the spot inoculation method, with an inoculation diameter not exceeding 5mm. After incubation at 37℃ for 48h, iodine solution was sprayed onto the medium. If the colony produced amylase, a clear zone would appear around the colony). PCR further verified that the expression cassette was integrated into the AmyE site of the Bacillus licheniformis strain, thus expressing the parental α-amylase.

[0122] Following the methods described in 3.1-3.2 above, Bacillus licheniformis integration plasmids and expression strains for each mutant (S000-S271) in Table 1 were constructed, with the difference being that the parental α-amylase gene was replaced with the gene of each mutant shown in Table 1. Table 1 shows the mutations of each mutant relative to the amino acid sequence shown in SEQ ID NO: 5 of the parent, where the amino acid positions correspond to the positions in SEQ ID NO: 1. Table 1. Mutant sites

[0123] Example 4: Shake-flask fermentation The *Bacillus licheniformis* strain expressing the parental α-amylase SEQ ID NO: 5 obtained in Example 3 and the α-amylase mutant strain shown in Table 1 were subjected to shake-flask fermentation as follows: A single activated bacterial clone was inoculated into 20 ml of culture medium (containing 4.0% maltose syrup, 2.0% peptone, 0.1% yeast extract, 0.6% KH2PO4, and corresponding antibiotics) and cultured to the logarithmic growth phase. 1.2 ml of the culture was inoculated into 30 ml of culture medium (containing 12.0% maltose syrup, 1.0% peptone, 1% yeast extract, 0.2% KH2PO4, and 0.003% MnCl2) and cultured in a reciprocating shaker at 220 rpm for 3 days. The fermentation broth was collected, and the amylase activity was measured. Example 5: Amylase Activity Assay Definition of amylase activity unit: 1 mL of liquid enzyme liquefies 1 mg of soluble starch in 1 minute under conditions of pH 6.0 and 70℃, which is 1 unit of enzyme activity, expressed as U / mL.

[0124] The percentage of heat-resistant residue refers to the percentage of enzyme activity after incubation at pH 4.0 and 100℃ for 30 min, relative to the enzyme activity before incubation (pH 6.0 and 70℃).

[0125] The enzyme activity assay method is as follows: Mix 20 ml of 20 g / L soluble starch solution with 5 ml of pH 6.0 phosphate buffer, preheat at 70℃ for 8 min, add 1.0 ml of diluted enzyme solution, react for 5 minutes, then take 1 ml of the reaction solution and add it to a test tube containing 0.5 ml of 0.1 mol / L hydrochloric acid solution and 5 ml of dilute iodine solution, shake well, and use 0.5 ml of 0.1 mol / L hydrochloric acid solution and 5 ml of dilute iodine solution as blanks. Measure the absorbance at 660 nm wavelength. According to Appendix B of GB / T 24401-2009 "National Food Safety Standard for Food Additives and Enzyme Preparations for Food Industry", absorbance and α... Using the amylase concentration reference table, the concentration of the test enzyme solution can be determined, and then the calculation formula can be applied. (Where X represents enzyme activity, C is the concentration of the test enzyme sample, N is the sample dilution factor, and 16.67 is the conversion factor calculated according to the definition of enzyme activity), to obtain the enzyme activity of the test sample.

[0126] Relative enzyme activity % refers to the enzyme activity of each amylase mutant relative to the parent, that is, the ratio (expressed as a percentage) of the enzyme activity of each mutant relative to the enzyme activity of the parent, which is set as 100%.

[0127] The tolerance factor is the ratio of the heat resistance residual percentage of the α-amylase mutant to the heat resistance residual percentage of the α-amylase mutant S000.

[0128] When an amylase with the amino acid sequence shown in SEQ ID NO: 5 is used as the parent, the thermostable residual % and relative enzyme activity % of the α-amylase mutant S000 are shown in Table 2.

[0129] Table 2: Thermostable Residue (%) and Relative Enzyme Activity (%) of α-Amylase Mutant S000

[0130] The tolerance factors of each mutant, and the relative enzyme activity % when the amylase shown in SEQ ID NO: 5 is used as the parent, are shown in Table 3.

[0131] Table 3. Tolerance factors and relative enzyme activity % of α-amylase mutants

[0132] The amino acid and nucleotide sequences involved in this invention (partial): SEQ ID NO: 1 AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVN PSDRNQEISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFF PDWLSYVRSQTGKPLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPCVFY GDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTINSDGWGEFKVNGGSVSVWVPRKTTVS SEQ ID NO: 2 Bacillus stearothermophilus wild-type α-amylase MLTFHRIIRKGWMFLLAFLLTASLFCPTGQPAKAAAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVNPSDRNQEISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGKPLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPCVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTINSDGWGEFKVNGGSVSVWVPRKTTVSTIARPITTRPWTGEFVRWTEPRLVAWP The nucleotide sequence encoding the wild-type α-amylase of Geobacillus stearothermophilus, SEQ ID NO: 3 SEQ ID NO: 4 is derived from Bacillus licheniformis ( 地衣芽孢杆菌 ) ANLNGTLMQYFEWYMPNDGQHWRRLQNDSAYLAEHGITAVWIPPAYKGTSQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKGGADATEDVTAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWHWYHFDGTDWDESRKLNRIYKFQGKAWDWEVSNENGNYDYLMYADIDYDHPDVAAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWQNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQR SEQ ID NO: 5 AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVNPSDRNQEISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGKAWDWEVDTEFGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGKPLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPCVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGGTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTINSDGWGEFKVNGGSVSVWVPRKTTVS SEQ ID NO: 6 GGTACCAGCTATTGTAACATAATCGGTACGGGGGTGAAAAAGCTAACGGAAAAGGGAGCGGAAAAGAATGATGTAAGCGTGAAAAATTTTTTATCTTATCACTTGAAATTGGAAGGGAGATTCTTTATTATAAGAAAACGGATGCTGAAGGAAGGAAACGAAGTCGGCAACCATTCCTGGGACCATCCGTTATTGACAAGGCTGTCAAATGAAAAAGCGTATCAGGAGATTAACGACACGCAAGAAATGATCGAAAAAATCAGCGGACACCTGCCTGTACACTTGCGTCCTCCATACGGCGGGATCAATGATTCCGTCCGCTCTCGCTCTTCCAATCTGAAGGTTTCATTGTGGGATGTTGATCCGGAAGATTGGAAGTACAAAAATAAGCAAAAGATTGTCAATCATGTCATGAGCCATGCGGGAGACGGAAAAATCG TCTTAATGCACGATATTTATGCAACGTCCGCAGATGCTGCTGAAGAGATTATTAAAAAGCTGAAAGCAAAAGGCTATCAATTGGTAACTGTATCTCAGCTTGAAGAAGTGAAGAAGCAGAGAGGCTATTGAATAAATGAGTAGAAAGCGCCATATCGGCGCTTTTCTTTTGGAAGAAAATATAGGGAAAATGGTATTTGTTAAAAATTCTGAATATTTATACAATATCATATGTTTCACAGGGAGGAGAATCGGCCTTAAGGGCCTGCAATCGATTGTTTGAGAAAAGAAGAAGACCATAAAAATACCTTGTCTGTCATCAGACAGGGTATTTTTTATGCTGTCCAGACTGTCCGCTGTGTAAAAAAAAGGAATAAAGGGGGGTTGACATTATTTTTTACTGATATGTATAATATAATTTGTATAAGAAAATGGAGCTC SEQ ID NO: 7 TCAATAATAATAACGCTGTGTGCTTTAAGCACACAGCGTTTTTTAGTGTGTATGAATCGAGATCCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCT SEQ ID NO: 8 ATGATTCAAAAACGAAAGCGGACAGTTTCGTTCAGACTTGTGCTTATGTGCACGCTGTTATTTGTCAGTTTGCCGATTACAAAAACATCAGCCGCA SEQ ID NO: 9 Bacillus licheniformis ( 地衣芽孢杆菌 ) amylase mutant VNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKAISQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKAGADATEDVTAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWYWYHFDGTDWDESRKLNRIYKFQGKTWDWEVSNEFGNYDYLMYADFDYDHPDVVAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWSNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQR SEQ ID NO: 10 derived from Bacillus subtilis ( 枯草芽孢杆菌 ) NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKGTSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVYADVVFNHKAGADGTEFVDAVEVDPSNRNQETSGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGTDWDESRKLNRIYKFRSTGKAWDWEVDTENGNYDYLMFADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYVRNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKSSGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAYAFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRDYIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYDLTGNRSDTVTINADGWGEFKVNGGSVSIWVAKTSNVTFTVNNATTTSGQNVYVVANIPELGNWNTANAIKMNPSSYPTWKATIALPQGKAIEFKFIKKDQAGNVIWESTSNRTYTVPFSSTGSYTASWNVP SEQ ID NO: 11 is derived from 噬纤维菌属 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTSQADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGDVVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTYSNFKWQWFHFDGTDWDQSRSLSRIFKFRGTGKAWDWEVSSENGNYDYLMYADIDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARAATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGGYYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAFILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQRDYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWYDLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ SEQ ID NO: 12 is derived from Bacillus halodurans ( Bacillus halmapalus ) HHNGTNGTMMQYFEWHLPNDGNHWNRLRDDASNLRNRGITAIWIPPAWKGTSQNDVGYGAYDLYDLGEFNQKGTVRTKYGTRSQLESAIHALKNNGVQVYGDVVMNHKGGADATENVLAVEVNPNNRNQEISGDYTIEAWTKFDFPGRGNTYSDFKWRWYHFDGVDWDQSRQFQNRIYKFRGDGKAWDWEVDSENGNYDYLMYADVDMDHPEVVNELRRWGEWYTNTLNLDGFRIDAVKHIKYSFTRDWLTHVRNATGKEMFAVAEFWKNDLGALENYLNKTNWNHSVFDVPLHYNLYNASNSGGNYDMAKLLNGTVVQKHPMHAVTFVDNHDSQPGESLESFVQEWFKPLAYALILTREQGYPSVFYGDYYGIPTHSVPAMKAKIDPILEARQNFAYGTQHDYFDHHNIIGWTREGNTTHPNSGLATIMSDGPGGEKWMYVGQNKAGQVWHDITGNKPGTVTINADGWANFSVNGGSVSIWVKR SEQ ID NO: 13 is derived from the genus Bacillus ( Bacillus sp. ) HHDGTNGTIMQYFEWNVPNDGQHWNRLHNNAQNLKNAGITAIWIPPAWKGTSQNDVGYGAYDLYDLGEFNQKGTVRTKYGTKAELERAIRSLKANGIQVYGDVVMNHKGGADFTERVQAVEVNPQNRNQEVSGTYQIEAWTGFNFPGRGNQHSSFKWRWYHFDGTDWDQSRQLANRIYKFRGDGKAWDWEVDTENGNYDYLMYADVDMDHPEVINELNRWGVWYANTLNLDGFRLDAVKHIKFSFMRDWLGHVRGQTGKNLFAVAEYWKNDLGALENYLSKTNWTMSAFDVPLHYNLYQASNSSGNYDMRNLLNGTLVQRHPSHAVTFVDNHDTQPGEALESFVQGWFKPLAYATILTREQGYPQVFYGDYYGIPSDGVPSYRQQIDPLLKARQQYAYGRQHDYFDHWDVIGWTREGNASHPNSGLATIMSDGPGGSKWMYVGRQKAGEVWHDMTGNRSGTVTINQDGWGHFFVNGGSVSVWVKR SEQ ID NO: 14 is derived from the genus Bacillus ( Bacillus sp. )) HHNGTNGTMMQYFEWYLPNDGNHWNRLRSDASNLKDKGISAVWIPPAWKGASQNDVGYGAYDLYDLGEFNQKGTIRTKYGTRNQLQAAVNALKSNGIQVYGDVVMNHKGGADATEMVRAVEVNPNNRNQEVSGEYTIEAWTKFDFPGRGNTHSNFKWRWYHFDGVDWDQSRKLNNRIYKFRGDGKGWDWEVDTENGNYDYLMYADIDMDHPEVVNELRNWGVWYTNTLGLDGFRIDAVKHIKYSFTRDWINHVRSATGKNMFAVAEFWKNDLGAIENYLNKTNWNHSVFDVPLHYNLYNASKSGGNYDMRQIFNGTVVQRHPMHAVTFVDNHDSQPEEALESFVEEWFKPLAYALTLTREQGYPSVFYGDYYGIPTHGVPAMKSKIDPILEARQKYAYGRQNDYLDHHNIIGWTREGNTAHPNSGLATIMSDGAGGNKWMFVGRNKAGQVWTDITGNRAGTVTINADGWGNFSVNGGSVSIWVNK SEQ ID NO: 15 Bacillus licheniformis ( Bacillus licheniformis ) amylase mutant VNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKAISQADVGYGAYDLYDLGEFWQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKAGADATEDVTAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWYWYHFDGTDWDESRKLNRIYLFQGKTWDWPVSNEFGNYDYLMYADYDYDYPDVVAEITRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWSNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPSVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGVSSVANSGLAALITDGPGGAKWMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQR SEQ ID NO: 16 Bacillus licheniformis ( Bacillus licheniformis ) amylase mutant VNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKAISQADVGYGAYDLYDLGEFWQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKAGADATEDVQAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWYWYHFDGTDWDESRKLNRIYLFQGKTWDWPVSNEFGNYDYLMYADYDYDYPDVVAEITRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWSNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPSVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGVSSVANSGLAALITDGPGGAKWMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQR SEQ ID NO: 17 Amylase Mutant of Geobacillus stearothermophilus AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGAYDLYDLGEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVNPSDRNQVISGTYQIQAWTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYLFEGKAWDWEVDTEFGNYDYLMYADLDMDHPEVTTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSSTGKPLFTVGEYWSGDINKLHNYITKTDGTVSLFDAPLHYKFYNASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPCVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGGTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTINSDGWGEFKVNGGSVSVWVPRKTTVSTIARP SEQ ID NO: 18 is derived from Microbacterium acetivorans ( Exiguobacterium acetylicum ) AGKATADNGTMMQYFEWYVPNDGNHWNRLGSDATKLDQLGITSVWIPPAYKGTSQNDVGYGAYDLYDLGEFNQKGTVRTKYGTKAQLKTAIGQLHTAGIDVYGDVVMNHKGGADFTEAVTAVEINPGNRNQEISGDYQIQAWTGFNFAARNNLYSNFKWKWYHFDGTDWDQSRSKSAIYKFRGTGKAWDTDVSTENGNYDYLMYADLDFDHPEVQQEMKNWGKWYVNELGLDGFRLDAVKHIKHGYLADWLANVRQTTGKPLFTVAEYWQNDLGTLQNYLSRTNYQQSVFDAPLHYKFEQASKGGGYYDMRTIFDGTLVKSNPVQAVTLVENHDSQPGQSLESTVQSWFKPLAYAMILTREQGYPSVFYGDYYGTKGTSNREIPALASKIDPLLKARKDFAFGKQNDYLDNQDIIGWTREGVSDRAKSGLATILSDGPGGSKWMYVGLQNKGEVWTDITGNNTASVTINQDGYGQFFVNGGSVSVYRQQ SEQ ID NO: 19 is derived from Exiguobacterium sibiricum ( Exiguobacterium sibiricum ) DNGTMMQYFEWYVPNDGNHWNRLGSDSTKLDQLGITSVWIPPAYKGTTQNDVGYGAYDLYDLGEFNQKGTVRTKYGTKAQLKTAINQLHTAGIDVYGDVVMNHKGGADFTEAVTAVEVNGSNRNQEISGDYQIQAWTGFDFAARNNTYSNFKWKWYHFDGTDWDQSRSKSAIYKFRGTGKAWDTDVSTENGNYDYLMYADIDFDHPEVQQEMKNWGKWYVNELGLDGFRLDAVKHIKHGYLADWLANVRQTTGKPLFTVAEYWQNDLGTLQNYLSRTNYQQSVFDAPLHYKFEQASKGGGYYDMRTIFDGTLVKTNPVQAVTLVENHDSQPGQSLESTVQSWFKPLAYAMILTREQGYPSVFYGDYYGTKGTSNREIPALASKIDPLLKARKDFAFGKQNDYLDNADVIGWTREGVTDRAKSGLATILSDGPGGSKWMYVGLQNKGEVWTDITGNNTASVTINQDGYGQFFVNGGSVSVYRQQ SEQ ID NO: 20 is derived from Paenibacillus Paenibacillus curdlanilyticus ) ADNGTIMQYFEWYLPNDGAHWNRLNNDAQNLKNVGITAVWIPPAYKGGSSADVGYGVYDTYDLGEFNQKGTVRTKYGTKSELISAVNNLHAKGIAVYGDVVLNHRMNADATELVDAVEVDPNNRNVETTSTYQIQAWTQYDFPGRGNTYSSFKWRWYHFDGVDWDQSRGLNRIYKLRGDGKDWDWEVDSEYGNYDYLMGADLDFNHPDVVNETKTWGKWFVNTVNLDGVRLDAVKHIKFDFMRDWVNNVRSTTGKNLFAVGEYWHYDVNKLNSYITKTNGTMSLFDVPLHFRFYDASNGGGGYDMRNLLNNTLMSSNPMKAVTFVENHDTQPTQALQSTVQSWFKPLAYATILTREQGYPCVFYGDYYGTSDGKISSYKPIMDKLLNARKVYAYGTQRDYFDHPDIVGWTREGDAAHAGSGLATLITDGPGGSKWMYVGTSKAGQVWTDKTGNRSGTVTIDANGWGNFWVNGGSVSVWAK SEQ ID NO: 21 is derived from Bacillus megaterium ( Bacillus megaterium ) MERNHTIMQFFEWHVPADGEHWQRLKELAPQLKEQGIDSVWIPPVTKGVSSEDNGYGVYDLYDLGEFDQKGTVRTKYGTKQELHEAIDACHNHGINVYVDIVMNHKAAADEKETFHVIEVDPMNRTEEISEPFEIEGWTKFTFEGRGDKYSSFKWNFNHFNGTDYDDKNGKEGVFRIAGENKSWNENVDQEFGNYDYLMFANIDYDHPEVREEMINWGKWLADTLQCDGYRLDAIKHINHDFIKEFAHELSSSQEKPFYFVGEFWNPELTACQEFLDVIDYQIDLFDVSLHYKLHEASQQGRDFDLTTIFDDTLVKTHPLNVVTFVDNHDSQPNESLESWVEDWFKQSAYALILLREDGYPCVFYGDYFGIGGEHPIEGKEKDISALLHVRYDKAYGQQDDYFDHPNTIGWVRHGVEEFEKSGCAVVMSNGEDGEKRMFVGEHRSGQTWIDFTNNREDQVVIEEDGYGQFPVNGGSVSVWAEA SEQ ID NO: 22 is derived from Alicyclobacillus sp. 18711 ( Alicyclobacillus Sp.18711 ) AFAGDNGTMMQYFEWYLPNDGTLWTKMGSDASHLKSIGITGVWFPPAYKGQSQSDVGYGVYDMYDLGEFNQKGTVRTKYGTKAQLQSAITSLHNNGIQAYGDVVLNHRMGADATETISAVEVNPSNRNQVTSGAYNISAWTDFEFPGRGNTYSSFKWHSYYFDGVDWDQSRQLSGKIYQIQGTGKAWDWEVDSENGNYDYLMGADIDYDHPDVQTEVKNWGKWFVNTLNLDGVRLDAVKHIKFDYMSSWLSSVKSTTGKSNLFAVGEYWNTSLGALENYENKTNWSMSLFDVPLHMNFQAAANGGGYYDMRNLLNNTMMKNHPIQAVTFVDNHDTEPGQALQSWVSDWFKPLAYATILTRQEGYPCVFYGDYYGIPSQSVSAKSTWLDKQLSARKSYAYGTQHDYLDNQDVIGWTREGDSAHAGSGLATVMSDGPGGSKTMYVGTAHAGQVFKDITGNRTDTVTINSAGNGTFPCNGGSVSIWVKQ SEQ ID NO: 23 is derived from Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) VNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVRTKYGTKSELQDAIGSLHSRNVQVYGDVVLNHKAGADATEDVTAVEVNPANRNQVTSEEYQIKAWTDFRFPGRGNTYSDFKWHWYHFDGADWDESRKISRIFKFRGEGKAWDWEVSSENGNYDYLMYADVDYDHPDVVAETKKWGIWYANELSLDGFRIDAAKHIKFSFLRDWVQAVRQATGKEMFTVAEYWQNNAGKLENYLNKTSFNQSVFDVPLHFNLQAASSQGGGYDMRRLLDGTVVSRHPEKAVTFVENHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGTSPKEIPSLKDNIEPILKARKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYAGLKNAGETWYDITGNRSDTVKIGSDGWGEFHVNDGSVSIYVQK SEQ ID NO: 24 is derived from the genus Clostridium ( Clostridium sp. ) MDNGLMFQGFEWYMPDDGNYYKDLKKKLVDMKRIGVTSVWLPPVCKATGSNDTGYGVYDLYDLGEFDQKGSVRTKYGTKEELLDLIKAIHDEGMYVYADVVLNHKAGADFEEEFMAVKVDN NNRTKEIEKQRNIKAWTGFNFPGNRNGKYSDFTWNYNHFSGVDYDASTGDKGIFRIIGENKGWNWGVSHDNGNFDYLMFADIDHANTEVKEELKRWVDWFIEELNLDGIRFDAVKHIDSAFLE EFTSHIKEKMGDEFYFLGEYWDHDVKNKIKFMKSTKYSMDLFDVGLHFNMYAASQNSANYDLRKLFDNTVTKTDPAMSVTFVDNHDSEPGQSLESFVKEWFKEIAYGIILLRKDGYPCIFY GDYYGIGGEFMIKPLKEKIDVLSLIRKNHAYGAQDDYFKEKDLIGWVRQGTEDHPGKCAVVISTREKKTISMFIDKYHSGKVYADFTGNCADKVKVDEEGYGEFTAEAGSISVWLEEEIVLG The embodiments of the present invention are not limited to those described above. Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and improvements to the present invention in form and detail, and these are all considered to fall within the protection scope of the present invention.

Claims

1. An α-amylase mutant containing 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, 273, 274, 276, 277, 278, 28. Substitution, insertion, and / or deletion at one or more positions of SEQ ID NO: 1, 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 positions correspond to SEQ ID NO: 1, 282, 284, 293, 294, 296, 297, 300, 302, 304, 305, 307, 309, 310, 311, 312, 313, 316, 317, 318, 319 ... The mutant is located at position 1 in SEQ ID NO: 1, wherein the mutant has at least 60% 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, SEQ ID NO: 9-24, and wherein the mutant has α-amylase activity.

2. The mutant of claim 1, comprising substitution, insertion, and / or deletion at one or more of the following positions: 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; 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. 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. 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; 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; 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; 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. 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. 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; 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. 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; 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; 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; 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. 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; 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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; 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; 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. 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; 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; 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; 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; 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; 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. 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. 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; 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. 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; 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. 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. 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; 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; 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. 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; 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; 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; 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; 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. 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; 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; 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. 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 D; 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. 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. 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. 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. 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; 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. 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; 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; 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. 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. 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; 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. 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; 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. 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. 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; 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; 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. 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. 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. 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; 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; 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; 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; 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; 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; 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. 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. 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; 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; 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; 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. 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; 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; 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; 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; 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. 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; 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; The amino acid at position 416 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; 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. 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; 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; 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; 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; The amino acid position thereon corresponds to the position in SEQ ID NO:

1.

3. The mutant of claim 1 or 2, comprising 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, E129 L, E129P, E129V, I130T, I130V, G132D, G132E, T133A, T133E, T133K, T133P, T133S, T133V, T133W, Y134E, Y134F, Q135D, Q135E, Q135G, Q135K, Q135M, Q1 35N, Q135P, Q135R, Q135T, Q135W, I136A, I136V, Q137A, Q137D, Q137K, A138 L, D143E, V164F, V164T, E168A, L172E, Y176F, Y176L, K177L, F178I, R179A, R 179E, R179K, R179S, A184E, A184K, A184Q, E188P, T191D, T191K, T191N, F19 3K, L204I, L204V, M206Y, H208N, V212T, F241Y, Q254S, S267T, N271K, L273Q , H274E, H274K, H274L, H274T, Y276F, I277L, T278E, T278K, N281D, G282D, G 282N, G282Y, M284I, M284V, N293Y, K294N, K294R, Y296A, T297E, T297K, T29 7N, K300N, G302N, A304G, A304K, A304N, A304S, F305Y, M307L, T309D, T309E , T309K, T309N, L310I, M311E, M311F, M311I, M311L, M311T, T312D, T312E, T 312K, T312N, N313D, N313G, M316A, M316L, M316T, M316V, K317E, K317R, K31 7S, D318E, D318K, D318L, D318T, D318Y, Q319A, Q319H, Q319K, Q319R, Q319Y,T321D, T321E, L322H, L322K, L322Y, E333Q, Q339E, W341Q, W341R, D343E, D343K, D343Q, F353L, F353Y, Q358N, Q358S, E359D, I372V, 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.

4. The mutant according to any one of claims 1-3, wherein the mutant has 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% and less than 100% 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, SEQ ID NO: 9-24 or its mature polypeptide.

5. The mutant according to any one of claims 1-4, comprising substitutions at one or more of the following amino acid positions: 59, 184, 188, 193, 254, 284, 293, 297 and / or 416; Preferably, the substitution at amino acid position 59 is 59A; The substitutions at amino acid position 184 are: 184E, 184Q, or 184K; The substitution at amino acid position 188 is: 188P; The substitution at amino acid position 193 is: 193K; The substitution at amino acid position 254 is: 254S; The substitution at amino acid position 284 is: 284V or 284I; The substitution at amino acid position 293 is: 293Y; and / or The substitution at amino acid position 416 is: 416S.

6. The mutant of claim 5, further comprising substitutions at one or more of the amino acid positions 122, 124, 128, 129, 133, 135, 191, 212, and 281; Preferably, the substitution at amino acid position 122 is: 122D; The substitutions at amino acid position 124 are: 124N, 124F, 124Y, or 124T; The substitution at amino acid position 128 is either 128K or 128E; The substitutions at amino acid position 129 are: 129I, 129V, 129K, 129A, 129L, or 129P; The substitutions at amino acid position 133 are: 133E, 133V, 133W, 133P, 133A, 133K, or 133S; The substitutions at amino acid position 135 are: 135E, 135K, 135D, 135T, 135N, 135P, 135W, 135G, 135M, or 135R. The substitutions at amino acid position 191 are: 191D, 191N, or 191K; The substitution at amino acid position 212 is: 212T; and / or The substitution at amino acid position 281 is: 281D.

7. The mutant of claim 5 or 6, comprising a group of substitutions selected from any one of the following: 59A / 188P / 254S / 293Y; 59A / 188P / 254S / 293Y / 212T; 59A / 188P / 254S / 293Y / 281D; and 59A / 188P / 254S / 293Y / 212T / 281D; Preferably, a group of substitutions selected from any one of the following: V59A / E188P / Q254S / N293Y; V59A / E188P / Q254S / N293Y / V212T; V59A / E188P / Q254S / N293Y / N281D; and V59A / E188P / Q254S / N293Y / V212T / N281D.

8. The mutant according to any one of claims 5-7, further comprising a substitution at amino acid position 179, preferably 179S, 179E, 179A or 179K.

9. The mutant of claim 8, further comprising substitutions at one or more of amino acid positions 129, 191, 212 and / or 281; Preferably, the substitution at amino acid position 129 is 129V or 129I; The substitution at amino acid position 191 is: 191N or 191D; The substitution at amino acid position 212 is: 212T; and / or The substitution at amino acid position 281 is: 281D.

10. The mutant of claim 8 or 9, wherein the amino acid sequence represented by SEQ ID NO: 5 comprises a group of substitutions selected from any one of the following: V59A / A184Q / E188P / Q254S / N293Y; V59A / A184Q / E188P / Q254S / N293Y / N281D / V212T; and V59A / A184Q / E188P / Q254S / N293Y / N281D / V212T / E129V / T297N / R179S / T191N / M284V.

11. The mutant according to any one of claims 8-10, wherein the amino acid sequence represented by SEQ ID NO: 5 comprises a group of substitutions selected from any one of the following: V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E18 8P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / I372V / Q374A / Y375K; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y / I372V / Q374A / Y375K; V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y / I372V / Q374A / Y375K; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304G / M311L / T312N / N313G / M316V / K317S / Q319H / I372V / Q374A / Y375K; V59A / N122D / S124N / Q128K / E129V / T133V / Q135K / Q137A / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316V / K317R / Q319Y; 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; 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; 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; 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; 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; 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; V59A / W115T / V119Y / N122D / S124N / Q128K / E129I / G132D / T133V / Q135P / Q137D / E168A / R179K / A184Q / E188P / T191N / F193K / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / W115K / V119Y / N122D / S124N / Q128K / E129I / G132E / T133V / Q135P / Q137D / E168A / R179K / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; V59A / W115K / D117L / V119Y / N122D / S124N / Q128K / E129I / G132D / T133V / Q135E / Q137D / E168A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; V59A / N122D / S124N / Q128K / G132D / T133V / Q135K / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117K / N122D / S124N / Q128K / E129V / G132D / T133E / Q135E / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / N122D / S124N / Q128K / G132E / T133V / Q135K / Q137A / Y176F / R179A / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117K / N122D / S124N / Q128K / G132E / T133V / Q135E / Q137A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117K / N122D / S124N / Q128K / E129I / G132E / T133V / Q135E / Q137A / Y176F / R179E / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274K / I277L / N281D / M284I / N293Y / T297E / A304K / F305Y / M311L / T312D / M316A / D318Y / Q319Y / T321D / L322K / S406K; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274E / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T312D / M316V / D318Y / Q319H / T321D / S406K; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / V212T / Q254S / S267T / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318T / Q319H / L322K; V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / S267T / N271K / H274E / I277L / T278E / M284I / N293Y / T297N / N313D / D318Y / Q319H / L322K; V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / S267T / H274L / I277L / T278K / M284I / N293Y / T297N / N313D / D318Y / Q319H; V59A / E129V / R179S / A184Q / E188P / V212T / Q254S / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318T / Q319H / L322K; V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / H274K / I277L / T278E / M284I / N293Y / T297N / N313D / D318Y / Q319Y / L322K; V59A / E129V / R179S / A184Q / E188P / T191D / V212T / Q254S / N271K / H274E / I277L / T278K / M284I / N293Y / T297N / N313D / D318Y / Q319H; 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; 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; V59A / D117K / N122D / S124N / Q128K / E129V / T133E / Q135E / Q137A / R179S / A184Q / E188P / T191N / V21 2T / Q254S / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / Q319Y / I372V / Q374A / Y375K; 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; 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; 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; 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; 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; 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; 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; 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; 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; T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204V / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N; T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204I / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N; T50A / V59A / F104V / G108A / E129V / D143E / R179S / A184Q / E188P / T191D / L204V / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N; T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204I / M206Y / H208N / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / A304N / F305Y / M311L / Q358S / E359D / I389K / S406K / G416S / A426V / Y450E / L452I; 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; 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; T50S / V59A / F104V / G108A / E129V / R179S / A184Q / E188P / T191D / L204I / M206Y / H208N / Q254S / N281D / M284 V / N293Y / T297N / A304K / F305Y / M311L / Q358N / E359D / I389K / S406K / G416S / A426V / T429S / Y450E / L452I; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N271K / H274E / T27 8E / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / D318Y / Q319H; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N271K / H274E / T278K / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / N313D / M316T / D318Y / Q319H; V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / H274L / T278E / N281D / M284V / N293Y / T297N / A304K / M311L / T312D / M316T / D318Y / Q319K; 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; D18A / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341R / I372V / Q374K / Y375F; D18A / T50E / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341R / I372V / Q374K; or D18A / R52Y / V59A / E129V / R179S / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N / E333Q / Q339E / W341Q / I372V / Q374A / Y375K.

12. The mutant according to any one of claims 5-7, further comprising substitutions at one or more positions of amino acid positions 177, 212 and / or 281, preferably 177L, 212T, and 281D, respectively.

13. The mutant of claim 12, further comprising substitutions at one or more of the amino acid positions 117, 122, 124, 128, 129, 133, 135, 176, and 191; Preferably, the substitution at amino acid position 117 is: 117E, 117T, 117W, 117M, 117S, 117L or 117K; The substitution at amino acid position 122 is: 122D; The substitutions at amino acid position 124 are: 124N, 124F, 124Y, or 124T; The substitution at amino acid position 128 is either 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 substitution at amino acid position 176 is: 176F or 176L; The substitution at amino acid position 191 is: 191D, 191N, or 191K.

14. The mutant of claim 12 or 13, wherein the amino acid sequence represented by SEQ ID NO: 5 comprises a group of substitutions selected from any one of the following: V59A / K177L / E188P / V212T / Q254S / N281D / N293Y; V59A / E129V / K177L / A184Q / E188P / T191N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / K177L / E188P / V212T / Q254S / N281D / N293Y / Y176F / N122D / A184E / Q128K / T191D; and V59A / K177L / E188P / V212T / Q254S / N281D / N293Y / Y176F / N122D / A184E / Q128K / T191D / S124N.

15. The mutant according to any one of claims 12-14, wherein the amino acid sequence represented by SEQ ID NO: 5 comprises a group of substitutions selected from any one of the following: V59A / E129I / L172E / Y176F / K177L / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / A118V / E129I / I136A / A138L / V164F / L172E / K177L / F178I / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / A118V / V119Q / D125N / E129I / I136A / A138L / V164F / L172E / Y176L / K177L / F178I / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N; V59A / V119Q / E129I / I136V / V164T / L172E / Y176F / K177L / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N; V59A / D125N / N127L / E129I / I136V / V164F / L172E / K177L / A184Q / E188P / T191D / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N; V59A / D125N / N127L / E129I / I136V / V164F / L172E / K177L / A184Q / E188P / H208N / V212T / F241Y / Q254S / N281D / M284V / N293Y / T297N; V59A / V119Q / E129I / I136V / V164T / L172E / Y176L / K177L / A184Q / E188P / T191N / H208N / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127R / Q128K / E129I / I130V / G132E / T133W / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / V119Y / N122D / S124N / D125N / N127L / Q128K / E129I / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274K / T278K / N281D / M284V / N293Y / T297N; 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; V59A / D117E / V119Q / N122D / S124N / Q128K / E129I / G132E / T133W / Q135K / Y176F / K177L / A184E / E188P / T191K / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N; V59A / D117L / N122D / S124N / Q128K / E129I / T133E / Q135W / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274K / T278K / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / V212T / Q254S / S267T / H274L / T278K / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / D125N / N127L / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / T278E / N281D / M284V / N293Y / T297N; V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274L / T278K / N281D / M284V / N293Y / T297N; V59A / D117L / N122D / S124N / Q128K / E129I / T133E / Q135W / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / T278K / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / V212T / Q254S / H274L / I277L / T278K / N281D / M284V / N293Y / T297N; V59A / E129V / Y176F / K177L / A184E / E188P / V212T / Q254S / H274K / I277L / T278K / N281D / M284V / N293Y / T297N / A304K / T309K / M311I / T312D / M316L / K317R / D318E / Q319H; V59A / E129V / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / H274L / T278E / N281D / M284V / N293Y / T297N / A304K / T309N / M311I / T312K / N313D / K317E / D318K / Q319K; V59A / E129V / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / H274K / T278K / N281D / M284V / N293Y / T297N / A304K / M311T / T312E / K317R / D318E / Q319H; 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; 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; 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; V59A / D117E / N122D / S124N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129L / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128E / E129K / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129P / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124Y / D125N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124T / D125N / N127L / Q128K / E129A / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124F / N127L / Q128K / E129K / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129P / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129P / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / Q128K / E129I / T133E / Q135E / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135T / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129I / T133P / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / T133V / Q135N / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124F / N127R / Q128K / E129I / T133P / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / D125N / Q128K / E129I / T133E / Q135P / Y176F / K177L / A184E / E188P / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117T / N122D / S124N / D125N / N127L / Q128E / E129L / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129I / T133V / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117S / N122D / S124N / Q128K / E129L / T133E / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127R / Q128K / E129V / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129A / I130V / G132D / T133W / Q13 5N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; V59A / D117E / N122D / S124N / N127R / Q128K / E129I / I130V / G132E / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / I130V / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / G132D / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V121M / N122D / S124N / N127L / Q128K / E129A / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129K / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V121M / N122D / S124N / D125N / N127L / Q128K / E129A / G132E / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / E120D / N122D / S124N / N127L / Q128K / E129K / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129V / I130V / G132E / T133W / Y134F / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127R / Q128K / E129L / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V119Q / N122D / S124N / D125N / N127L / Q128K / E129P / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / N127L / Q128K / E129K / G132E / T133W / Q135D / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; V59A / D117T / N122D / S124N / N127R / Q128K / E129I / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117W / V119Q / N122D / S124N / N127L / Q128K / E129I / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / G132E / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / V119Y / N122D / S124N / D125N / N127L / Q128K / E129I / I130T / G132D / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / D125N / N127L / Q128K / E129K / I130V / G132D / T133W / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V119Q / N122D / S124N / D125N / N127L / Q128K / E129I / I130V / G132D / T133W / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117M / N122D / S124N / D125N / N127L / Q128K / E129K / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V119Y / N122D / S124N / N127L / Q128K / E129I / I130V / G132E / T133A / Q135N / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117W / V119Y / E120D / N122D / S124N / Q128K / E129I / G132D / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / V119Y / N122D / S124N / Q128K / E129I / I130T / G132D / T133A / Q135T / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124F / N127R / Q128K / E129I / G132D / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117W / V119Y / E120A / N122D / S124N / Q128K / E129I / I130T / G132E / T133E / Y134E / Q135K / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117W / E120D / N122D / S124N / N127L / Q128K / E129L / I130V / G132E / T133E / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132D / T133W / Q135D / Y176F / K177L / A184K / E188P / T191D / V212T / Q254S / N281D / M284V / N293Y / T297N; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; 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; V59A / D117E / N122D / S124N / Q128K / E129I / T133V / Q135E / Y176F / K177L / A184E / E188P / T191D / V212T / Q25 4S / H274K / N281D / M284I / N293Y / T297E / A304K / M311L / T312E / M316L / D318E / Q319Y / T321E / D343E / S406K; 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; 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; 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 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.

16. The mutant according to any one of claims 5-7, further comprising substitutions at one or more of the following amino acid positions: 117, 122, 124, 128, 129, 130, 132, 133, 135, 176, 177, 191, 212, 274, 277, 281, 304, 311, 312, 318, 319, 321, and 406, preferably at these positions. The substitutions are 117E, 122D, 124N, 128K, 129I, 130V, 132E, 133W, 135E, 176F, 177L, 191D, 212T, 274E, 274K, 277L, 281D, 304K, 311L, 312D, 312E, 312K, 318E, 318Y, 319H, 319Y, 321D and / or 406K.

17. The mutant of claim 16, further comprising a substitution at amino acid position 316, preferably 316T.

18. The mutant of claim 16 or 17, wherein the amino acid sequence represented by SEQ ID NO: 5 comprises a group of substitutions selected from any one of the following: 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; and 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.

19. The mutant according to any one of claims 16-18, wherein the amino acid sequence represented by SEQ ID NO: 5 comprises a group of substitutions selected from any one of the following: 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; V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / 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; V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / 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; V59A / D117E / N122D / S124N / Q128K / E129I / I130V / G132E / T133W / Q135E / 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 / ; 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 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.

20. The mutant according to any one of claims 5-7, further comprising substitutions at one or more of the following amino acid positions: 117, 122, 124, 127, 128, 129, 133, 135, 176, 177, 191, 212, 274, 277, 281, 304, 311, 312, 318, 319, 321, and 406, preferably, these... The substitutions at the positions are 117E, 122D, 124N, 127R, 128K, 129I, 133V, 135K, 176F, 177L, 191D, 212T, 274K, 274E, 277L, 281D, 304K, 311L, 312D, 312K, 318Y, 318E, 319H, 319Y, 321D and / or 406K.

21. The mutant of claim 20, further comprising substitutions at amino acid positions 316, 343 and / or 359, preferably 316T, 343E or 343K, 359D, respectively.

22. The mutant of claim 20 or 21, wherein the amino acid sequence represented by SEQ ID NO: 5 comprises a group of substitutions selected from any one of the following: V59A / D117E / N122D / S124N / N127R / Q128K / E129I / T133V / Q135K / Y176F / K177L / A184E / E188P / T191D / V212T / Q254S / I277L / N281D / M284I / N293Y / T297E / A304K / M311L / T321D / S406K; and 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.

23. The mutant according to any one of claims 20-22, wherein the amino acid sequence represented by SEQ ID NO: 5 comprises a group of substitutions selected from any one of the following: 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; 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; 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; 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 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.

24. A polynucleotide encoding an α-amylase mutant according to any one of claims 1-23.

25. A recombinant expression vector comprising the polynucleotide of claim 24.

26. A host cell comprising the polynucleotide of claim 24 or the recombinant expression vector of claim 25.

27. The host cell as described in claim 26, wherein it is a bacterium.

28. The host cell of claim 27, wherein the bacteria are derived from the genus Bacillus.

29. The host cell as described in any one of claims 26-28, wherein it is Bacillus subtilis or Bacillus licheniformis.

30. A method for preparing the α-amylase mutant according to any one of claims 1-23, comprising the following steps: (a) Culture the host cells according to any one of claims 26-29 under conditions suitable for the expression of the α-amylase mutant; (b) Recover the expressed α-amylase mutant.

31. An α-amylase mutant obtained by the method of claim 30.

32. A composition comprising the α-amylase mutant according to any one of claims 1-23 and one or more other enzymes.

33. The composition of claim 32, 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.

34. Use of the α-amylase mutant according to any one of claims 1-23 or the composition according to any one of claims 32-33 for liquefying starch-containing materials.

35. Use of the α-amylase mutant according to any one of claims 1-23 or the composition according to any one of claims 32-33 for washing.

36. Use of the α-amylase mutant of any one of claims 1-23 or the composition of any one of claims 32-33 for desizing textiles.

37. Use of the α-amylase mutant of any one of claims 1-23 or the composition of any one of claims 32-33 for producing baked goods.

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