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

Abstract

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

Description

α-Amylase mutants, their preparation methods and applications

[0001] Cross-citation of related applications

[0002] This application claims priority to Chinese patent application CN 202411999621.5, filed on December 31, 2024, which is incorporated herein by reference in its entirety. Technical Field

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

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

[0005] In various industries such as starch sugar, citric acid, and alcohol, starch is first liquefied by amylase and then saccharified by saccharifying enzymes to produce glucose for subsequent production. However, amylase is easily inactivated above 100℃ or below pH 5.0, while the jet liquefaction temperature in the starch liquefaction process can reach above 110℃, and the optimal pH for the subsequent saccharification step is around 4.5. Therefore, the starch sugar industry requires α-amylases that are heat-resistant, acid-resistant, or have enhanced enzyme activity. The α-amylase BLA (Bacillus licheniformis α-amylase, SEQ ID NO:1) from Bacillus licheniformis has attracted widespread attention due to its relatively excellent heat and acid resistance. Novozymes disclosed in its international patent application WO2002 / 010355A2 a mutant LE399 (SEQ ID NO:15) of a hybrid α-amylase composed of the N-terminal 1-37 amino acid residues of α-amylase from Bacillus amyloliquefaciens and the C-terminal 40-483 amino acid residues of α-amylase from Bacillus licheniformis. Compared to the wild-type BLA, LE399 exhibits improved stability under high temperature and low pH conditions. However, there is still a need for more novel α-amylases with improved properties, namely α-amylases with higher activity and / or greater stability under high temperature and low pH conditions. Summary of the Invention

[0006] The present invention aims to provide an α-amylase mutant that, compared with parental α-amylase and / or wild-type α-amylase and / or other α-amylase mutants, has improved enzyme activity, thermal stability and / or low pH stability, preferably with improved stability under high temperature and low pH conditions, and the amino acid positions described herein correspond to the amino acid positions of SEQ ID NO:1.

[0007] In one aspect, the present invention provides an α-amylase mutant comprising amino acid positions 48, 49, 52, 68, 101, 111, 114, 116, 118, 123, 124, 126, 127, 128, 131, 132, 133, 134, 136, 140, 142, 148, 152, 156, 167, 168, 169, 170, 171, 172, 173, 175, 178, 181, 188, 19 Insertion, deletion, and / or substitution at one or more of the following positions: 1, 205, 265, 271, 272, 275, 278, 279, 280, 281, 283, 291, 294, 298, 301, 302, 307, 309, 312, 314, 315, 316, 318, 319, 320, 338, 340, 356, 372, 373, 374, 375, 406, wherein the amino acid position corresponds to the position in SEQ ID NO:1, wherein the mutant has at least 60% and less than 100% sequence identity with any of the amino acid sequences shown in SEQ ID NO:1-15 or their mature polypeptides, and wherein the mutant has α-amylase activity.

[0008] In some embodiments, the α-amylase mutant of the present invention comprises substitutions at two, three, or four positions selected from amino acid positions 156, 205, 265, and 320; and the mutant does not contain any combination of substitutions selected from H205Y / Y156W and H205C / H156Y. In some embodiments, the substitution at amino acid position 156 is 156H or 156T; the substitution at amino acid position 205 is 205D; the substitution at amino acid position 265 is 265G; and / or the substitution at amino acid position 320 is 320A.

[0009] In some embodiments, the α-amylase mutant of the present invention comprises substitutions at one, two, three, four, or five positions of amino acid positions 123, 127, 128, 134, and 181. In some embodiments, the substitution at amino acid position 123 is 123F, 123L, 123N, 123T, or 123Y; the substitution at amino acid position 127 is 127E or 127K; the substitution at amino acid position 128 is 128A, 128D, 128E, 128I, 128K, 128L, or 128P; the substitution at amino acid position 134 is 134D, 134E, 134K, 134M, 134N, 134P, 134T, or 134W; and / or the substitution at amino acid position 181 is 181E.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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

[0014] 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.

[0015] 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.

[0016] SEQ ID NO:1 is Bacillus licheniformis α-amylase.

[0017] SEQ ID NO:2 is Bacillus licheniformis α-amylase mutant M35.

[0018] SEQ ID NO:3 is Bacillus α-amylase TS-23 described in the Journal of Applied Microbiology, 1997, 82:325-334 (SWALL:q59222).

[0019] SEQ ID NO:4 is the α-amylase AMY1048 of Bacillus flavothermus described in WO2005 / 001064.

[0020] SEQ ID NO:5 is Bacillus α-amylase TS-22 described as SEQ ID NO:21 in WO04 / 113511.

[0021] SEQ ID NO:6 is Bacillus amyloliquefaciens α-amylase.

[0022] SEQ ID NO:7 is the Bacillus alkaline species SP690 amylase described in WO95 / 26397 as SEQ ID NO:1.

[0023] SEQ ID NO:8 is the Bacillus halmapalus α-amylase described in WO95 / 26397 as SEQ ID NO:2.

[0024] SEQ ID NO:9 is the Bacillus alkaline species AA560 amylase described in WO00 / 60060 as SEQ ID NO:4.

[0025] SEQ ID NO:10 is the Bacillus alkaline species A7-7 amylase described in WO200210356 as SEQ ID NO:2.

[0026] SEQ ID NO:11 is the Bacillus basic species SP707 amylase described in Tsukamoto et al., 1988, Biochem. Biophys. Res. Commun. 151:25-33.

[0027] SEQ ID NO:12 is the Bacillus alkaline species K-38 amylase described as SEQ ID NO2 in EP1022334.

[0028] SEQ ID NO:13 is the Bacillus licheniformis α-amylase described in Lee et al., 2006, J. Biochem, 139:997-1005.

[0029] SEQ ID NO:14 is α-amylase from Geobacillus stearothermophilus.

[0030] SEQ ID NO:15 is a variant α-amylase LE399 previously disclosed, for example, in WO2002 / 010355A2.

[0031] When the terms “about” or “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%).

[0032] 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."

[0033] 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.

[0034] 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.

[0035] 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 means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; insertion means adding one or more 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-amylase activity), but may have variations in activity levels. For example, a mutant may remain unchanged or improve upon at least one activity or property (e.g., α-amylase activity) relative to the parent polypeptide.

[0036] The term "parent" refers to a polypeptide that undergoes changes to produce a mutant, and 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, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. s) α-Amylase, or Gram-negative bacterial polypeptides, such as α-amylase or variants derived from Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma. For example, the parent can be α-amylase or variants derived from Bacillus licheniformis, Bacillus flavus, Bacillus amyloliquefaciens, Bacillus salivarius, or Bacillus stearothermophilus.

[0037] The parent can be a naturally occurring (wild-type) polypeptide or a mutant thereof prepared by any suitable means. For example, the parent protein can be a mutant of a naturally occurring polypeptide whose amino acid sequence has been modified or altered. Thus, the parent α-amylase may have one or more amino acid substitutions, deletions, and / or insertions. Thus, the parent α-amylase may be a mutant of the wild-type α-amylase. The term “parent” as used herein can include, for example, the mutant LE399 mentioned herein, and can also include mutants containing the V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W mutation, such as the mutant M35 described herein. As used herein, the term "parent" may include, for example, any of the α-amylases in SEQ ID NO:1-15, or any α-amylase having at least 60% sequence identity with any of the polypeptides in SEQ ID NO:1-15. The term "parent" as used herein may also include polypeptides containing fragments of any of SEQ ID NO:1-15.

[0038] 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:1-15, 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:1-15 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide).

[0039] 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.

[0040] 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 temperatures and / or low pH) is higher than that of other α-amylase mutants and / or parental α-amylases and / or wild-type α-amylases.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The term "control sequence" refers to the nucleic acid 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.

[0049] 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.

[0050] 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.

[0051] The term "sequence consistency" describes the correlation between two amino acid sequences or two nucleotide sequences. Sequence consistency refers to the percentage of sequence similarity determined by performing optimal alignment (maximum sequence consistency) of two sequences within a comparison window. Optimal alignment can be achieved through additions or deletions (i.e., gaps). The sequence consistency percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs under optimal alignment mode to generate the number of matching positions, dividing the number of matching positions by the total number of positions compared, and then multiplying the result by 100 to obtain the sequence consistency percentage. Sequence consistency between two amino acid sequences can be determined using available local alignment tools (e.g., BLAST) or global alignment tools (e.g., the Needleman-Wunsch algorithm). For example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) can be used to determine sequence identity between two amino acid sequences. The parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). Alternatively, the parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL substitution matrix (the EMBOSS version of NCBI NUC4.4).

[0052] "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 the reference amino acid sequence (e.g., the optimal alignment described above). In this invention, the positions of amino acids in the described α-amylase mutants are all determined based on the amino acid sequence shown in SEQ ID NO:1, a reference amino acid sequence. The identification of corresponding amino acid residues in another α-amylase can be determined by comparing multiple peptide sequences using several computer programs with their respective default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparison by logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acids Research, 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research, 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research, 33:511-518; Katoh and Toh, 2007, Bioinformatics, 23:372-374; Katoh et al., 2009, Methods in Molecular Biology, 537:39-64; Katoh and Toh, 2010, Bioinformatics, 26:1899-1900) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research, 22:4673-4680).

[0053] When other α-amylases are dissimilar to the polypeptide of SEQ ID NO:1, making conventional sequence-based comparisons ineffective in detecting their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), alternative pairwise sequence comparison algorithms can be used. Higher sensitivity in sequence-based searches can be achieved using search programs that utilize the probabilistic representations (spectrums) of polypeptide families to search a database. For example, the PSI-BLAST program generates multiple spectra through an iterative database search process and can detect distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even higher sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics. 19: 874-881) utilize multiple sources (PSI-BLAST, secondary structure prediction, structure alignment spectra, and solvation potential) Information about the potential is used as input to a neural network that predicts the structural folding of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using a variety of 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, Proteins. 33: 88-96). (Engineering.11:739-747) compares two or more protein structures, and implementations of these algorithms can also be used to query a structure database with the target structure in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics.16:566-567).

[0054] The mutant representation method of this invention: Amino acids are represented by conventional single-letter or three-letter names. As is known to those skilled in the art, the single-letter names for amino acids are as follows: A for alanine, corresponding to the three-letter name Ala; C for cysteine, corresponding to the three-letter name Cys; D for aspartic acid, corresponding to the three-letter name Asp; E for glutamic acid, corresponding to the three-letter name Glu; F for phenylalanine, corresponding to the three-letter name Phe; G for glycine, corresponding to the three-letter name Gly; H for histidine, corresponding to the three-letter name His; I for isoleucine, corresponding to the three-letter name Ile; K for lysine, corresponding to the three-letter name Lys; L for leucine, ... The three letters corresponding to these three letters are named Leu; M is methionine, corresponding to Met; N is asparagine, corresponding to Asn; P is proline, corresponding to Pro; Q is glutamine, corresponding to Gln; R is arginine, corresponding to Arg; S is serine, corresponding to Ser; T is threonine, corresponding to Thr; V is valine, corresponding to Val; W is tryptophan, corresponding to Trp; and Y is tyrosine, corresponding to Tyr.

[0055] In this document, the following nomenclature is used: original amino acid, position, existing amino acid. For example, an amino acid substitution can be represented by the form "Y156H", where 156 indicates that the substitution occurs at the 156th amino acid corresponding to the reference sequence (e.g., SEQ ID NO:1), the Y preceding 156 is the amino acid that occupied that position before the substitution, and the H following 156 is the amino acid that occupied that position after the substitution. When an amino acid change is represented by the form "V3(AAPF)", it means that the amino acid at that position was V before the change and is AAPF after the change. 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, "156H" can represent that any amino acid at position 156 is substituted with H, or that amino acid H is present at position 156; "3(AAPF)" can represent that any amino acid at position 3 is substituted with AAPF (SEQ ID NO:28), or that AAPF (SEQ ID NO:28) is present at position 3. Multiple mutations are connected by " / ", for example, "Y156H / Y205D / N265G / S320A" means that amino acid Y at position 156 is replaced with H, amino acid Y at position 205 is replaced with D, amino acid N at position 265 is replaced with G, and amino acid S at position 320 is replaced with A. On the other hand, "156H / 205D / 265G / 320A" means that any amino acid at position 156 is replaced with H, any amino acid at position 205 is replaced with D, any amino acid at position 265 is replaced with G, and any amino acid at position 320 is replaced with A; or the amino acid at position 156 is H, the amino acid at position 205 is D, the amino acid at position 265 is G, and the amino acid at position 320 is A.

[0056] 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.

[0057] This invention relates to α-amylase mutants possessing α-amylase activity, thermal stability, and / or low pH stability, particularly stability at high temperatures and low pH. In some embodiments, the α-amylase mutants of this invention exhibit increased α-amylase activity and / or increased thermal stability and / or increased low pH stability compared to parental α-amylase and / or wild-type α-amylase and / or other α-amylase mutants. In some embodiments, the parental α-amylase may be the amino acid sequence shown in SEQ ID NO:2 (referred to herein as mutant M35), which has the amino acid alterations V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W relative to the α-amylase mutant LE399, and exhibits improved α-amylase activity, thermostability, and / or low pH stability relative to mutant LE399. In some embodiments, the parental α-amylase may be wild-type BLA (SEQ ID NO:1). In some embodiments, the parental α-amylase may be mutant LE399 (SEQ ID NO:15). In some embodiments, the parental α-amylase may be any one of the α-amylases in SEQ ID NO:3-14. In this invention, the positions of the amino acids in the described α-amylase mutants are determined based on the amino acid sequence shown in SEQ ID NO:1, a reference amino acid sequence.

[0058] In some embodiments, the α-amylase mutants of the present invention can exhibit significantly improved amylase activity, 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.

[0059] In some embodiments, the α-amylase mutant of the present invention comprises amino acid alterations relative to the parental α-amylase, which will be described in detail below. In some embodiments, the parental α-amylase may be mutant M35, such as the α-amylase shown in SEQ ID NO:2. In some embodiments, the parental α-amylase may be wild-type BLA, such as the α-amylase shown in SEQ ID NO:1. In some embodiments, the parental α-amylase may be mutant LE399, such as the α-amylase shown in SEQ ID NO:15. In some embodiments, the parental α-amylase may be the α-amylase shown in any one of SEQ ID NO:3-14. In some embodiments, the parental α-amylase may be a mature polypeptide (e.g., a mature polypeptide of M35, wild-type BLA, or LE399) that does not contain a signal peptide. In some embodiments, the parental α-amylase may be a mature polypeptide described in any one of SEQ ID NO:1-15. In some embodiments, the parental α-amylase comprises a signal peptide (e.g., M35, wild-type BLA, or LE399 containing a signal peptide, such as a polypeptide obtained by adding a signal peptide to the N-terminus of SEQ ID NO:2, SEQ ID NO:1, or SEQ ID NO:15). In some embodiments, the parental α-amylase comprises a polypeptide obtained by adding a signal peptide to the N-terminus of any of the polypeptides described in SEQ ID NO:3-14.

[0060] 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:2). In some embodiments, the α-amylase mutant contains only the listed amino acid changes relative to the parental sequence (e.g., SEQ ID NO:2) and does not contain any amino acid changes at unlisted positions.

[0061] 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 the 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; when the parental α-amylase has different amino acids at that position, referring to an α-amylase mutant containing an amino acid change at that position also includes the case where the different amino acid is mutated into the mutated amino acid.

[0062] In some embodiments, the α-amylase mutant of the present invention comprises amino acid positions 48, 49, 52, 68, 101, 111, 114, 116, 118, 123, 124, 126, 127, 128, 131, 132, 133, 134, 136, 140, 142, 148, 152, 156, 167, 168, 169, 170, 171, 172, 173, 175, 178, 181, 18 The mutant has substitutions at one or more of the following positions: 8, 191, 205, 265, 271, 272, 275, 278, 279, 280, 281, 283, 291, 294, 298, 301, 302, 307, 309, 312, 314, 315, 316, 318, 319, 320, 338, 340, 356, 372, 373, 374, 375, and 406, wherein the amino acid positions correspond to the positions in SEQ ID NO:1, wherein the mutant has at least 60% and less than 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:1-15 or its mature polypeptide, and wherein the mutant has α-amylase activity.

[0063] 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 or more.

[0064] 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:2, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity. In some embodiments, the α-amylase mutant of the present invention is sequence-aligned with the amino acid sequence shown in SEQ ID NO:2 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide).

[0065] 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:1-15, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity. 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:1-15 within the amino acid sequence range of a mature polypeptide (e.g., a polypeptide sequence with or without a signal peptide).

[0066] 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 have an amino acid sequence as shown in SEQ ID NO:18.

[0067] In some embodiments, the α-amylase mutant of the present invention comprises substitutions at one or more of the following positions:

[0068] The amino acid at position 48 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S;

[0069] The amino acid at position 49 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A, V or Y, more preferably A;

[0070] The amino acid at position 52 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or S;

[0071] The amino acid at position 68 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0072] The amino acid at position 101 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0073] The amino acid at position 111 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S;

[0074] The amino acid at position 114 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by F, L, M, E or T, more preferably by E or T or by F, L or M.

[0075] The amino acid at position 116 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by N or Q or D or E or K or L or M or R or W, more preferably by E, R, D, M, K, W or L or D, E, K, L, M, N or Q or D, E, K, L, M, N, Q or R;

[0076] The amino acid at position 118 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably by Q;

[0077] The amino acid position 123 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V. Preferably, it is replaced by E or H or I or K or Q or R or V or F or L or N or T or Y. More preferably, it is replaced by N, F, Y, L or T or by E, H, I, K, N, Q, R, T, V or Y or by E, F, H, I, K, N, L, Q, R, T, V or Y.

[0078] The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0079] The amino acid at position 126 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C or L, more preferably C.

[0080] The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by H or Q or E or R or D or M or K or W or L, more preferably by E, R, D, M, K, W or L or by E, H, K or Q, more preferably by E or K;

[0081] The amino acid at position 128 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. Preferably, it is replaced by M, Q, R, T, A, D, E, I, K, L, or P. More preferably, it is replaced by P, L, K, I, E, D, or A, or by A, D, E, I, K, M, P, Q, R, or T, or by A, D, E, I, K, L, or P.

[0082] The amino acid at position 131 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D, E or K.

[0083] The amino acid at position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or T or V or A or K or P or W, more preferably by W, P, A or K, or by D, K, P, T or V or by D, K, P, T, V or W;

[0084] The amino acid at position 133 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F or Y, more preferably Y;

[0085] The amino acid position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or F or D or E or K or M or N or P or T or W, more preferably D or E or K or M or N or P or T or W.

[0086] The amino acid at position 136 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A, D or E, more preferably A;

[0087] The amino acid at position 140 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or R, more preferably R;

[0088] The amino acid at position 142 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably D;

[0089] The amino acid at position 148 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or T or N, more preferably by D or N or D or T, and even more preferably by N.

[0090] The amino acid at position 152 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S;

[0091] The amino acid at position 156 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by H or T or F, more preferably by H or T or by F or H.

[0092] The amino acid at position 167 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0093] The amino acid at position 168 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K;

[0094] The amino acid at position 169 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably T;

[0095] The amino acid at position 170 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N;

[0096] The amino acid at position 171 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K;

[0097] 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 or K or L or W, more preferably K.

[0098] The amino acid at position 173 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0099] The amino acid at position 175 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F or W, more preferably F;

[0100] The amino acid at position 178 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or K, more preferably K;

[0101] The amino acid at position 181 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by T or A or D or E or G or N or R or S, more preferably by E.

[0102] The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by E or G or N or S or D or K or R or T, more preferably by T, R, K or D or by E, G, K, N, S or T.

[0103] The amino acid at position 191 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C;

[0104] The amino acid at position 205 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or N, more preferably by D;

[0105] The amino acid at position 265 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably G;

[0106] The amino acid at position 271 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or L, more preferably L;

[0107] The amino acid at position 272 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R;

[0108] The amino acid at position 275 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E;

[0109] The amino acid at position 278 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E or G or K, more preferably by K;

[0110] The amino acid at position 279 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y.

[0111] The amino acid at position 280 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V;

[0112] The amino acid at position 281 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C or F or I or L or T or V or M, more preferably M.

[0113] The amino acid at position 283 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0114] The amino acid at position 291 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or R.

[0115] The amino acid position 294 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E or K or L or N or Q or R or S or T or V or Y, more preferably by Q, N or E.

[0116] The amino acid at position 298 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or S.

[0117] The amino acid position 301 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or E or H or L or N or Q or R or S or T or V or Y or K, more preferably K.

[0118] The amino acid at position 302 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. Preferably, it is replaced by A, G, L, V, W, or F. More preferably, it is replaced by F.

[0119] The amino acid at position 307 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L or I.

[0120] The amino acid at position 309 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R or A or H or Q or D or E, more preferably by E or D.

[0121] The amino acid at position 312 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0122] The amino acid at position 314 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0123] The amino acid at position 315 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or T or V, more preferably T or V.

[0124] The amino acid position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or E or F or L or M or Q or R or V or W or E or Y, more preferably Y.

[0125] The amino acid position 318 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by Q or D or E, more preferably by E or D.

[0126] The amino acid at position 319 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L;

[0127] The amino acid at position 320 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A;

[0128] The amino acid at position 338 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably P, Q or R.

[0129] The amino acid at position 340 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or K or M or N or R or A or E or P, more preferably by A, E or P.

[0130] The amino acid at position 356 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E;

[0131] The amino acid at position 372 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably P or S.

[0132] The amino acid at position 373 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by G, L or T.

[0133] The amino acid at position 374 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or S.

[0134] The amino acid at position 375 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or K or N or T.

[0135] The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or D or P or R or K, more preferably K.

[0136] The amino acid position thereon corresponds to the position in SEQ ID NO:1.

[0137] In some embodiments, the α-amylase mutant of the present invention comprises one or more amino acids selected from the group consisting of: 48S, 49A, 49V, 49Y, 52D, 52S, 68N, 101L, 111S, 114F, 114L, 114M, 114E, 114T, 116N, 116Q, 116D, 116E, 116K, 116L, 116M, 116R, 116W, 118Q, 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123F, 123L, 123N, 123T, 123Y, 124N, 126C, 126L, 127E, 127K, 128M, 128Q, 1 28R, 128T, 128A, 128D, 128E, 128I, 128K, 128L, 128P, 131D, 131E, 131K, 132 D. 132T, 132V, 132A, 132K, 132P, 132W, 133F, 133Y, 134A, 134F, 134Q, 134S, 1 34V, 134D, 134E, 134K, 134M, 134N, 134P, 134T, 134W, 136A, 136D, 136E, 140 K, 140R, 142D, 148D, 148T, 148N, 152S, 156H, 156T, 156F, 167Y, 168K, 169T, 1 70N, 171K, 172E, 172L, 172W, 172K, 173A, 175F, 175W, 178E, 178K, 181T, 181 A. 181D, 181G, 181N, 181R, 181S, 181E, 188E, 188G, 188N, 188S, 188D, 188K, 188R, 188T, 191C, 205D, 205N, 265G, 271K, 271L, 272R, 275E, 278D, 278E, 27 8G, 278K, 279Y, 280V, 281C, 281F, 281I, 281L, 281T, 281V, 281M, 283L, 291K, 291R, 294D, 294L, 294R, 294S, 294T, 294V, 294Y, 294E, 294K, 294N, 294Q, 29 8A, 298S, 301A, 301E, 301H, 301L, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y, 301K, 302A, 302G, 302L, 302V, 302W, 302F, 307L, 307I, 309R, 309A, 309H, 30 9Q, 309D, 309E, 312L, 314A, 315E, 315T, 315V, 316A, 316E, 316F, 316L, 316M,316Q, 316R, 316V, 316W, 316E, 316Y, 318Q, 318D, 318E, 319L, 320A, 338P, 338Q, 338R, 340D, 340K, 340M, 340N, 340R, 340A, 340E, 340P, 356D, 356E, 372P, 372S, 373G, 373L, 373T, 374D, 374S, 375D, 375K, 375N, 375T, 406A, 406D, 406P, 406R, and 406K.

[0138] In some embodiments, the α-amylase mutant of the present invention comprises one or more amino acids selected from the group consisting of: 48S, 49A, 52D, 52S, 68N, 101L, 111S, 114E, 114T, 116D, 116E, 116K, 116L, 116M, 116R, 116W, 118Q, 123F, 123L, 123N, 123T, 123Y, 124N, 126C, 126L, 127E, 127K, 128A, 128D, 1 28E, 128I, 128K, 128L, 128P, 131D, 131E, 131K, 132A, 132K, 132P, 132W, 133F, 133Y, 134D, 134E, 134K, 134M, 1 34N, 134P, 134T, 134W, 136A, 136D, 136E, 140K, 140R, 142D, 148D, 148T, 152S, 156H, 156T, 167Y, 168K, 169T, 17 0N, 171K, 172K, 173A, 175F, 178E, 178K, 181E, 188D, 188K, 188R, 188T, 191C, 205D, 265G, 271K, 271L, 272R, 27 5E, 278D, 278E, 278G, 278K, 279Y, 280V, 281M, 283L, 291K, 291R, 294E, 294K, 294N, 294Q, 298A, 298S, 301K, 302 F, 307I, 309D, 309E, 312L, 314A, 315E, 315T, 315V, 316Y, 318D, 318E, 319L, 320A, 338P, 338Q, 338R, 340A, 340E, 340P, 356D, 356E, 372P, 372S, 373G, 373L, 373T, 374D, 374S, 375D, 375K, 375N, 375T, 406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:1.

[0139] In some embodiments, the α-amylase mutant of the present invention comprises one or more substitutions selected from the group consisting of: 48S, 49A, 49V, 49Y, 52D, 52S, 68N, 101L, 111S, 114F, 114L, 114M, 114E, 114T, 116N, 116Q, 116D, 116E, 116K, 116L, 116M, 116R, 116W, 118Q, 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123F, 123L, 123N, 123T, 123Y, 124N, 126C, 126L, 127E, 127K, 128M, 128Q, 12 8R, 128T, 128A, 128D, 128E, 128I, 128K, 128L, 128P, 131D, 131E, 131K, 132D , 132T, 132V, 132A, 132K, 132P, 132W, 133F, 133Y, 134A, 134Q, 134S, 134V, 1 34F, 134D, 134E, 134K, 134M, 134N, 134P, 134T, 134W, 136A, 136D, 136E, 140 K, 140R, 142D, 148D, 148T, 148N, 152S, 156H, 156T, 156F, 167Y, 168K, 169T, 1 70N, 171K, 172E, 172L, 172W, 172K, 173A, 175F, 175W, 178E, 178K, 181T, 181 A. 181D, 181G, 181N, 181R, 181S, 181E, 188E, 188G, 188N, 188S, 188D, 188K, 188R, 188T, 191C, 205D, 205N, 265G, 271K, 271L, 272R, 275E, 278D, 278E, 27 8G, 278K, 279Y, 280V, 281C, 281F, 281I, 281L, 281T, 281V, 281M, 283L, 291K, 291R, 294D, 294L, 294R, 294S, 294T, 294V, 294Y, 294E, 294K, 294N, 294Q, 29 8A, 298S, 301A, 301E, 301H, 301L, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y, 301K, 302A, 302G, 302L, 302V, 302W, 302F, 307L, 307I, 309R, 309A, 309H, 30 9Q, 309D, 309E, 312L, 314A, 315E, 315T, 315V, 316A, 316E, 316F, 316L, 316M,316Q, 316R, 316V, 316W, 316E, 316Y, 318Q, 318D, 318E, 319L, 320A, 338P, 338Q, 338R, 340D, 340K, 340M, 340N, 340R, 340A, 340E, 340P, 356D, 356E, 372P, 372S, 373G, 373L, 373T, 374D, 374S, 375D, 375K, 375N, 375T, 406A, 406D, 406P, 406R, and 406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:1.

[0140] In some embodiments, the α-amylase mutant of the present invention comprises one or more substitutions selected from the group consisting of: 48S, 49A, 52D, 52S, 68N, 101L, 111S, 114E, 114T, 116D, 116E, 116K, 116L, 116M, 116R, 116W, 118Q, 123F, 123L, 123N, 123T, 123Y, 124N, 126C, 126L, 127E, 127K, 128A, 128D, 12 8E, 128I, 128K, 128L, 128P, 131D, 131E, 131K, 132A, 132K, 132P, 132W, 133F, 133Y, 134D, 134E, 134K, 134M, 13 4N, 134P, 134T, 134W, 136A, 136D, 136E, 140K, 140R, 142D, 148D, 148T, 152S, 156H, 156T, 167Y, 168K, 169T, 170 N, 171K, 172K, 173A, 175F, 178E, 178K, 181E, 188D, 188K, 188R, 188T, 191C, 205D, 265G, 271K, 271L, 272R, 275 E, 278D, 278E, 278G, 278K, 279Y, 280V, 281M, 283L, 291K, 291R, 294E, 294K, 294N, 294Q, 298A, 298S, 301K, 302F The amino acid positions are 307I, 309D, 309E, 312L, 314A, 315E, 315T, 315V, 316Y, 318D, 318E, 319L, 320A, 338P, 338Q, 338R, 340A, 340E, 340P, 356D, 356E, 372P, 372S, 373G, 373L, 373T, 374D, 374S, 375D, 375K, 375N, 375T, and 406K, where the amino acid positions correspond to the positions in SEQ ID NO:1.

[0141] In some embodiments, the α-amylase mutant of the present invention comprises one or more substitutions selected from the group consisting of: A48S, I49A, I49V, I49Y, A52D, A52S, W68N, V101L, A111S, W114F, W114L, W114M, W114E, W114T, T116N, T116Q, T116D, T116E, T116K, T116L, T116M, T116R, T116W, V118Q, A123E, A123H, A123I, A123K, A123Q, A123R, A123V, A123F, A123L, A123N, A123T, A123Y, D 124N, N126C, N126L, R127E, R127K, V128M, V128Q, V128R, V128T, V128A, V12 8D, V128E, V128I, V128K, V128L, V128P, G131D, G131E, G131K, E132D, E132T, E132V, E132A, E132K, E132P, E132W, H133F, H133Y, R134A, R134F, R134Q, R1 34S, R134V, R134D, R134E, R134K, R134M, R134N, R134P, R134T, R134W, K136A , K136D, K136E, H140K, H140R, H142D, S148D, S148T, S148N, D152S, Y156H, Y 156T, Y156F, E167Y, S168K, R169T, K170N, L171K, R172E, R172L, R172W, R17 2K, R173A, Y175F, Y175W, Q178E, Q178K, T181T, T181A, T181D, T181G, T181N , T181R, T181S, T181E, P188E, P188G, P188N, P188S, P188D, P188K, P188R, P1 88T, G191C, Y205D, Y205N, N265G, E271K, E271L, N272R, N275E, N278D, N278 E. N278G, N278K, F279Y, N280V, H281C, H281F, H281I, H281L, H281T, H281V, H 281M, V283L, Q291K, Q291R, A294D, A294L, A294R, A294S, A294T, A294V, A29 4Y, A294E, A294K, A294N, A294Q, Q298A, Q298S, G301A, G301E, G301H, G301L,G301N, G301Q, G301R, G301S, G301T, G301V, G301Y, G301K, Y302A, Y302G, Y302L, Y302V, Y302W, Y302F, L307L, L307I, N309R, N309A, N30 9H, N309Q, N309D, N309E, V312L, S314A, K315E, K315T, K315V, H316A, H316E, H316F, H316L, H316M, H316Q, H316R, H316V, H316W, H316E, H The amino acid sequences 316Y, L318Q, L318D, L318E, K319L, S320A, T338P, T338Q, T338R, Q340D, Q340K, Q340M, Q340N, Q340R, Q340A, Q340E, Q340P, S356D, S356E, D372P, D372S, S373G, S373L, S373T, Q374D, Q374S, R375D, R375K, R375N, R375T, H406A, H406D, H406P, H406R, and H406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:1. In some embodiments, the α-amylase mutant of the present invention includes the above substitutions relative to the amino acid sequence shown in SEQ ID NO:2. ,

[0142] In some embodiments, the α-amylase mutant of the present invention comprises one or more substitutions selected from the group consisting of: A48S, I49A, A52D, A52S, W68N, V101L, A111S, W114E, W114T, T116D, T116E, T116K, T116L, T116M, T116R, T116W, V118Q, A123F, A123L, A123N, A123T, A123Y, D124N, N126C, N126L, R127E, R127K, V128A, V128D, V128E. V128I, V128K, V128L, V128P, G131D, G131E, G131K, E132A, E132K, E132P, E132W, H133F, H133Y, R134D, R134E, R134K, R134M, R134N , R134P, R134T, R134W, K136A, K136D, K136E, H140K, H140R, H142D, S148D, S148T, D152S, Y156H, Y156T, E167Y, S168K, R169T, K170N , L171K, R172K, R173A, Y175F, Q178E, Q178K, T181E, P188D, P188K, P188R, P188T, G191C, Y205D, N265G, E271K, E271L, N272R, N275 E. N278D, N278E, N278G, N278K, F279Y, N280V, H281M, V283L, Q291K, Q291R, A294E, A294K, A294N, A294Q, Q298A, Q298S, G301K, Y302 F, L307I, N309D, N309E, V312L, S314A, K315E, K315T, K315V, H316Y, L318D, L318E, K319L, S320A, T338P, T338Q, T338R, Q340A, Q340E, Q340P, S356D, S356E, D372P, D372S, S373G, S373L, S373T, Q374D, Q374S, R375D, R375K, R375N, R375T, H406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:1. In some embodiments, the α-amylase mutant of the present invention includes the above substitutions relative to the amino acid sequence shown in SEQ ID NO:2.

[0143] In some embodiments, the α-amylase mutant of the present invention may contain amino acid substitutions as described below (1), (2), (3), or (4) or any combination thereof, or may include amino acid substitutions as described below (1), (2), (3), and (4):

[0144] (1) Within the area of ​​region 3, within the area of ​​region 4, and within and around region 6. Amino acid changes within the range, including amino acid substitutions at at least five positions selected from amino acid positions 116-136 (e.g., amino acid substitutions at positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, e.g., amino acid substitutions at positions 5-10, 5-7 or 6-10);

[0145] (2) Amino acid substitutions at at least two positions selected from amino acid positions 175-195 (e.g., amino acid substitutions at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 positions, e.g., amino acid substitutions at 2-4 positions or 3-4 positions);

[0146] (3) Amino acid substitutions at at least 5 positions selected from amino acid positions 296-320 (e.g., amino acid substitutions at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 positions, e.g., amino acid substitutions at 5-8 positions or 6-7 positions);

[0147] (4) Amino acid substitutions at any 1 to 7 positions selected from amino acid positions 271, 274, 281, 294, 340, 356 and 406 (e.g., amino acid substitutions at 1, 2, 3, 4, 5, 6 or 7 positions, e.g., amino acid substitutions at 4-7, 5-7 or 4-6 positions);

[0148] The specific amino acid substitutions at each of the aforementioned positions may be as described above and / or as described below.

[0149] In some embodiments, the α-amylase mutant of the present invention may contain amino acid substitutions at one or more or all of the following amino acid positions: 116, 123, 126, 128, 132, 156, 181, 265, 278, 281, 301, 309, 316, 318, 320, 340, 356, and 406. In some embodiments, based on any amino acid or amino acid substitution or combination thereof described in this paragraph, the α-amylase mutant of the present invention may further contain amino acid substitutions at one or more or all of the following amino acid positions: 127, 191, 205, and 271. In some embodiments, based on any amino acid or amino acid substitution or combination thereof described in this paragraph, the α-amylase mutant of the present invention may further contain amino acid substitutions at one or more or all of the following amino acid positions: 134, 175, 294, and 302. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further include amino acid substitutions at one or more or all of the following positions: 23, 39, 49, 61, 62, 74, 106, 114, 124, 133, 136, 140, 142, 148, 152, 154, 155, 172, 178, 188, 212, 213, 242, 274, 276, 306, 308, 313, 315, 370, 407, 417, 456. The specific amino acid substitutions at each of these positions may be, for example, as described above and / or as described below.

[0150] In some embodiments, the α-amylase mutant of the present invention may contain amino acid substitutions at one or more or all of the following amino acid positions: 116, 123, 126, 128, 132, 156, 181, 191, 265, 271, 278, 281, 301, 309, 316, 318, 320, 340, 356, and 406. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further contain amino acid substitutions at one or more or all of the following amino acid positions: 127, 175, and 205. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further contain amino acid substitutions at one or more or all of the following amino acid positions: 134, 294, and 302. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further include an amino acid substitution at the following amino acid position: 308. In some embodiments, based on any of the amino acids or amino acid substitutions or combinations thereof described in this paragraph, the α-amylase mutant of the present invention may further include an amino acid substitution at one or more or all of the following amino acid positions: 23, 39, 49, 61, 62, 74, 106, 114, 148, 154, 155, 172, 188, 212, 213, 242, 274, 276, 306, 313, 370, 407, 417, 456. The specific amino acid substitutions at each of these positions may be, for example, as described above and / or as described below.

[0151] In some embodiments, amino acid position 23 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably with M, Q or S.

[0152] In some embodiments, amino acid position 39 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with C or T.

[0153] In some embodiments, the amino acid position 61 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by M.

[0154] In some embodiments, amino acid position 62 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with W.

[0155] In some embodiments, amino acid position 74 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with A.

[0156] In some embodiments, amino acid position 106 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with L.

[0157] In some embodiments, amino acid position 154 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by L, R, T, or Y.

[0158] In some embodiments, the amino acid position 155 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably Y.

[0159] In some embodiments, the amino acid position 212 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with L.

[0160] In some embodiments, the amino acid position 213 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with K.

[0161] In some embodiments, amino acid position 242 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with A, E, F, L, N, S, or T.

[0162] The amino acid at position 274 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by I, M, or V.

[0163] In some embodiments, amino acid position 276 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with S.

[0164] The amino acid at position 306 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably by H, R, or Y.

[0165] The amino acid at position 308 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably by F or M.

[0166] The amino acid at position 313 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y or V, preferably L.

[0167] In some embodiments, amino acid position 370 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with H, Q, or R.

[0168] In some embodiments, amino acid position 407 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with E or R.

[0169] In some embodiments, amino acid position 417 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with P.

[0170] In some embodiments, amino acid position 456 is replaced with A, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y, or V, preferably with L or V.

[0171] In some embodiments, the α-amylase mutant of the present invention may contain substitutions at two, three, or four positions selected from amino acid positions 156, 205, 265, and / or 320. In some embodiments, the α-amylase mutant of the present invention does not contain a combination of substitutions selected from any of the following:

[0172] H205Y / Y156W;

[0173] H205C / H156Y.

[0174] In some embodiments, the α-amylase mutant of the present invention further does not contain any combination of substitutions or amino acid combinations selected from any of the following:

[0175] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 247Y / 360C / 416V / 437W / 126C / 191C / 205D / 31S / 265G;

[0176] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 126C / 191C / 156H / 148N / 265G;

[0177] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 213T / 247Y / 360C / 416V / 437W / 126C / 191C / 156H / 148N / 205D / 31S;

[0178] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 126C / 191C / 320A / 148N / 31S / 265G;

[0179] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 247Y / 360C / 416V / 437W / 126C / 191C / 320A / 148N / 205D;

[0180] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 126C / 191C / 320A / 156H / 31S;

[0181] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 213T / 247Y / 360C / 416V / 437W / 126C / 191C / 320A / 156H / 205D / 265G;

[0182] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 126C / 191C / 265G / 31S;

[0183] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 126C / 191C / 265G;

[0184] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 247Y / 360C / 416V / 437W / 126C / 191C / 148N / 205D / 31S / 265G;

[0185] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 320A;

[0186] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 156H;

[0187] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 265G;

[0188] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 320A / 181Q / 265G / 148N;

[0189] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 320A / 181Q / 49K;

[0190] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 49K / 265G / 148N;

[0191] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 474P / 49K / 320A / 265G;

[0192] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 474P / 181Q / 265G;

[0193] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 247Y / 360C / 416V / 437W / 474P / 320A / 148N;

[0194] 3(AAPF) / 68W / 114W / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 123N / 142T / 291N / 482P / 134E / 148N / 320A;

[0195] 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W / 49K / 181Q / 148N / 320A.

[0196] In some embodiments, the α-amylase mutant of the present invention further does not contain any combination of substitutions selected from the following:

[0197] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H247Y / Q360C / D416V / R437W / N126C / G191C / H205D / H31S / N265G;

[0198] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / N126C / G191C / Y156H / S148N / N265G;

[0199] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / K213T / H247Y / Q360C / D416V / R437W / N126C / G191C / Y156H / S148N / H205D / H31S;

[0200] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / N126C / G191C / S320A / S148N / H31S / N265G;

[0201] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H247Y / Q360C / D416V / R437W / N126C / G191C / S320A / S148N / H205D;

[0202] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / N126C / G191C / S320A / Y156H / H31S;

[0203] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / K213T / H247Y / Q360C / D416V / R437W / N126C / G191C / S320A / Y156H / H205D / N265G;

[0204] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / N126C / G191C / N265G / H31S;

[0205] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / N126C / G191C / N265G;

[0206] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H247Y / Q360C / D416V / R437W / N126C / G191C / S148N / H205D / H31S / N265G;

[0207] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / S320A;

[0208] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / Y156H;

[0209] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / N265G;

[0210] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / S320A / T181Q / N265G / S148N;

[0211] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / S320A / T181Q / I49K;

[0212] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / I49K / N265G / S148N;

[0213] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / G474P / I49K / S320A / N265G;

[0214] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / G474P / T181Q / N265G;

[0215] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / H247Y / Q360C / D416V / R437W / G474P / S320A / S148N;

[0216] V3(AAPF) / H68W / D114W / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / A123N / H142T / Q291N / Q482P / L134E / S148N / S320A;

[0217] V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W / I49K / T181Q / S148N / S320A.

[0218] In some embodiments, the α-amylase mutant of the present invention may contain two, three, or four substitutions selected from the group consisting of: a substitution at amino acid position 156 of 156F, 156H, or 156T; a substitution at amino acid position 205 of Y205D or 205N; a substitution at amino acid position 265 of 265G; and / or a substitution at amino acid position 320 of 320A. In some embodiments, the α-amylase mutant of the present invention may contain two, three, or four amino acids selected from the group consisting of 156F, 156H, or 156T, 205D, or 205N, 265G, and 320A. In some embodiments, the α-amylase mutant of the present invention may contain two, three, or four substitutions selected from the group consisting of: a substitution at amino acid position 156 of Y156F, Y156H, or Y156T; a substitution at amino acid position 205 of Y205D or Y205N; a substitution at amino acid position 265 of N265G; and / or a substitution at amino acid position 320 of S320A.

[0219] In some embodiments, the α-amylase mutant of the present invention may contain two, three, or four substitutions selected from the group consisting of: a substitution at amino acid position 156 of 156H or 156T; a substitution at amino acid position 205 of Y205D; a substitution at amino acid position 265 of 265G; and / or a substitution at amino acid position 320A. In some embodiments, the α-amylase mutant of the present invention may contain two, three, or four amino acids selected from the group consisting of: 156H or 156T, 205D, 265G, and 320A. In some embodiments, the α-amylase mutant of the present invention may contain two, three, or four substitutions selected from the group consisting of: a substitution at amino acid position 156 of Y156H or Y156T; a substitution at amino acid position 205 of Y205D; a substitution at amino acid position 265 of N265G; and / or a substitution at amino acid position 320A.

[0220] In some embodiments, the α-amylase mutant of the present invention does not contain one or more amino acids selected from the group consisting of: amino acids at amino acid position 265 being 265A, 265S, 265T, 265V, 265Y, 265C, 265Q, 265E, 265H, 265I, 265L, 265M, 265F, 265P, or 265W; amino acids at amino acid position 156 being 156Y, 156A, 156C, 156Q, 156E, 156G, 156I, 156L, 156M, 156P, 156W, etc. 6S, 156T, 156W, or 156V; amino acids at amino acid position 205 are 205C, 205A, 205G, 205I, 205L, 205M, 205F, 205P, 205W, 205Y, or 205V; amino acids at amino acid position 320 are 320R, 320N, 320D, 320C, 320Q, 320E, 320G, 320H, 320I, 320L, 320K, 320M, 320F, 320P, 320S, 320T, 320W, 320Y, or 320V.

[0221] In some embodiments, the α-amylase mutant of the present invention does not contain one or more substitutions selected from the group consisting of: substitutions at amino acid position 265 of 265A, 265S, 265T, 265V, 265Y, 265C, 265Q, 265E, 265H, 265I, 265L, 265M, 265F, 265P, or 265W; and substitutions at amino acid position 156 of 156Y, 156A, 156C, 156Q, 156E, 156G, 156I, 156L, 156M, 156P, 156W. 6S, 156T, 156W, or 156V; substitutions at amino acid position 205 are 205C, 205A, 205G, 205I, 205L, 205M, 205F, 205P, 205W, 205Y, or 205V; substitutions at amino acid position 320 are 320R, 320N, 320D, 320C, 320Q, 320E, 320G, 320H, 320I, 320L, 320K, 320M, 320F, 320P, 320S, 320T, 320W, 320Y, or 320V.

[0222] 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 include substitutions at one, two, three, four, or five positions of amino acid positions 123, 127, 128, 134, and 181; 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 one or more substitutions selected from the group consisting of: substitutions at amino acid position 123 such as 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123F, 123L, 123N, 123T, or 123 Y; substitutions at amino acid position 127 are: 127H, 127Q, 127E or 127K; substitutions at amino acid position 128 are: 128M, 128Q, 128R, 128T, 128A, 128D, 128E, 128I, 128K, 128L or 128P; substitutions at amino acid position 134 are: 134A, 134F, 134Q, 134S, 134V, 134D, 134E, 134K, 134M, 134N, 134P, 134T or 134W; and / or substitutions at amino acid position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S or 181E.

[0223] 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 include substitutions at one, two, three, four, or five positions of amino acid positions 123, 127, 128, 134, and 181; 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 one or more substitutions selected from the group consisting of: at amino acid position 123 The substitutions are: 123F, 123L, 123N, 123T or 123Y; the substitution at amino acid position 127 is: 127E or 127K; the substitution at amino acid position 128 is: 128A, 128D, 128E, 128I, 128K, 128L or 128P; the substitution at amino acid position 134 is: 134D, 134E, 134K, 134M, 134N, 134P, 134T or 134W; and / or the substitution at amino acid position 181 is: 181E.

[0224] 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 acids at position 123 being: 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123F, 123L, 123N, 123T, or 123Y; amino acids at position 127 being: 127H, 127Q, 127E, or 127K; amino acids at position 128 being... The amino acids at amino acid position 134 are: 134A, 134F, 134Q, 134S, 134V, 134D, 134E, 134K, 134M, 134N, 134P, 134T, or 134W; and / or the amino acids at amino acid position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E.

[0225] 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 one or more amino acids selected from the group consisting of: amino acid at position 123 being 123F, 123L, 123N, 123T, or 123Y; amino acid at position 127 being 127E or 127K; amino acid at position 128 being 128A, 128D, 128E, 128I, 128K, 128L, or 128P; amino acid at position 134 being 134D, 134E, 134K, 134M, 134N, 134P, 134T, or 134W; and / or amino acid at position 181 being 181E.

[0226] In some embodiments, the α-amylase mutant of the present invention may comprise a group of substitutions selected from any one of the following or a group of amino acids selected from any one of the following: 156H / 320A; 156H / 205D; 156H / 265G; 205D / 265G; 205D / 320A; 265G / 320A; 156H / 205D / 265G; 156H / 205D / 320A; 205D / 265G / 320A; 156H / 205D / 265G / 320A; 156H / 205D / 265G / 320A / 127K; 156H / 205D / 265G / 320A / 127K / 123N; 156H / 205D / 265G / 320A / 127K / 181E; and 156H / 205D / 265G / 320A / 127K / 123N / 181E.

[0227] In some embodiments, the α-amylase mutant of the present invention may comprise a group of substitutions selected from any one of the following: Y156H / S320A; Y156H / Y205D; Y156H / N265G; Y205D / N265G; Y205D / S320A; N265G / S320A; Y156H / Y205D / N265G; Y156H / Y205D / S320A; Y205D / N265G / S320A; Y1 56H / Y205D / N265G / S320A; Y156H / Y205D / N265G / S320A / R127K; Y156H / Y205D / N265G / S320A / R127K / A123N; Y156H / Y205D / N265G / S320A / R127K / T181E; and Y156H / Y205D / N265G / S320A / R127K / A123N / T181E.

[0228] 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 116, 123, 124, 126, 127, 128, 133, 134, 136, 140, 142, 148, 152, 178 and / or 181. 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 116 of 116D, 116K, 116L, 116N, 116Q, 116E, or 116M; a substitution at amino acid position 123 of 123E, 123H, 123I, 123K, 123Q, 123R, 123T, 123V, 123Y, or 123N; a substitution at amino acid position 124 of 124N; a substitution at amino acid position 126 of 126L or 126C; a substitution at amino acid position 127 of 127H, 127Q, 127K, or 127E; and a substitution at amino acid position 128 of 128I, 128K, 128M, 128Q, or 128R. 128T, 128E, 128A, 128D, or 128P; substitution at amino acid position 133: 133Y; substitution at amino acid position 134: 134A, 134F, 134Q, 134S, 134T, 134V, 134W, 134E, 134N, 134P, 134K, 134D, or 134M; substitution at amino acid position 136: 136A; substitution at amino acid position 14... The substitution at position 0 is 140R; the substitution at position 142 is 142D; the substitution at position 148 is 148D or 148N; the substitution at position 152 is 152S; the substitution at position 178 is 178K; and / or the substitution at position 181 is 181T, 181A, 181D, 181G, 181N, 181R, 181S or 181E.

[0229] 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 of the following positions: amino acid positions 116, 123, 124, 126, 127, 128, 133, 134, 136, 140, 142, 148, 152, 178, and / or 181. 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 116 as 116E or 116M; substitution at amino acid position 123 as 123N; substitution at amino acid position 124 as 124N; substitution at amino acid position 126 as 126L; substitution at amino acid position 127 as 127K or 127E; and substitution at amino acid position 128 as 128E, 128A, 128D, or... 128P; substitution at amino acid position 133: 133Y; substitution at amino acid position 134: 134E, 134N, 134P, 134K, 134D or 134M; substitution at amino acid position 136: 136A; substitution at amino acid position 140: 140R; substitution at amino acid position 142: 142D; substitution at amino acid position 148: 148D; substitution at amino acid position 152: 152S; substitution at amino acid position 178: 178K; and / or substitution at amino acid position 181: 181E.

[0230] 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 116 being 116D, 116K, 116L, 116N, 116Q, 116E, or 116M; amino acid at amino acid position 123 being 123E, 123H, 123I, 123K, 123Q, 123R, 123T, 123V, 123Y, or 123N; amino acid at amino acid position 124 being 124N; amino acid at amino acid position 126 being 126L or 126C; amino acid at amino acid position 127 being 127H, 127Q, 127K, or 127E; and amino acid at amino acid position 128 being 128I, 128K, 128M, 128Q, 128R, or 128M. 128T, 128E, 128A, 128D, or 128P; the amino acid at position 133 is 133Y; the amino acids at position 134 are 134A, 134F, 134Q, 134S, 134T, 134V, 134W, 134E, 134N, 134P, 134K, 134D, or 134M; the amino acid at position 136 is 136A; the amino acid at position 140... The amino acid at position 140 is 140R; the amino acid at position 142 is 142D; the amino acid at position 148 is 148D or 148N; the amino acid at position 152 is 152S; the amino acid at position 178 is 178K; and / or the amino acid at position 181 is 181T, 181A, 181D, 181G, 181N, 181R, 181S or 181E.

[0231] 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 116E or 116M at amino acid position 116; amino acid 123N at amino acid position 123; amino acid 124N at amino acid position 124; amino acid 126L at amino acid position 126; amino acid 127K or 127E at amino acid position 127; and amino acid 128E, 128A, 128D, or... 128P; amino acid at position 133 is 133Y; amino acid at position 134 is 134E, 134N, 134P, 134K, 134D, or 134M; amino acid at position 136 is 136A; amino acid at position 140 is 140R; amino acid at position 142 is 142D; amino acid at position 148 is 148D; amino acid at position 152 is 152S; amino acid at position 178 is 178K, and / or amino acid at position 181 is 181E.

[0232] 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 132, 175, 188, 281, 294, 301, 302, 309, 316, 318, 340, 356 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 substitutions selected from the group consisting of: substitutions at amino acid position 132 such as 132D, 132K, 132T, 132V, 132W or 132P; substitutions at amino acid position 175 such as 175F or 175W; and substitutions at amino acid position 188 such as 188E, 188G, 188N ... 8S, 188K, or 188T; substitutions at amino acid position 281 are: 281C, 281F, 281I, 281L, 281T, 281V, or 281M; substitutions at amino acid position 294 are: 294D, 294E, 294L, 294R, 294S, 294T, 294V, 294Y, 294N, or 294Q or 294K; substitutions at amino acid position 301 are: 301A, 301E, 301H, 301L, 301N. Substitutions at amino acid position 302 include: 302A, 302G, 302L, 302V, 302W, or 302F; substitutions at amino acid position 309 include: 309R, 309A, 309H, 309Q, 309D, or 309E; substitutions at amino acid position 316 include: 316A, 316E, 316F, 316L, 316M, 316Q, or 316R. The substitutions at amino acid position 318 are: 318E, 318Q, or 318D; the substitutions at amino acid position 340 are: 340A, 340D, 340K, 340M, 340N, 340R, 340E, or 340P; the substitutions at amino acid position 356 are: 356D or 356E; and / or the substitutions at amino acid position 406 are: 406A, 406D, 406P, 406R, or 406K.

[0233] 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 132, 175, 188, 281, 294, 301, 302, 309, 316, 318, 340, 356 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 substitutions selected from the group consisting of: substitution at amino acid position 132 as 132W or 132P; substitution at amino acid position 175 as 175F; substitution at amino acid position 188 as 188K or 188T; substitution at amino acid position 281 as 281M; and substitution at amino acid position 294 as 294N, or... 294Q or 294K; substitution at amino acid position 301: 301K; substitution at amino acid position 302: 302F; substitution at amino acid position 309: 309D or 309E; substitution at amino acid position 316: 316Y; substitution at amino acid position 318: 318D; substitution at amino acid position 340: 340E or 340P; substitution at amino acid position 356: 356D; and / or substitution at amino acid position 406: 406K.

[0234] 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 132 being 132D, 132K, 132T, 132V, 132W, or 132P; amino acid at amino acid position 175 being 175F or 175W; and amino acid at amino acid position 188 being 188E, 188G, 188N, or 132P. 88S, 188K, or 188T; amino acids at position 281 are 281C, 281F, 281I, 281L, 281T, 281V, or 281M; amino acids at position 294 are 294D, 294E, 294L, 294R, 294S, 294T, 294V, 294Y, 294N, 294Q, or 294K; amino acids at position 301 are 301A, 301E, 301H, 301L, or 301N. 301Q, 301R, 301S, 301T, 301V, 301Y, or 301K; amino acids at position 302 are 302A, 302G, 302L, 302V, 302W, or 302F; amino acids at position 309 are 309R, 309A, 309H, 309Q, 309D, or 309E; amino acids at position 316 are 316A, 316E, 316F, 316L, 316M, 316Q, or 316R. 316V, 316W, 316E, or 316Y; amino acids at amino acid position 318 are 318E, 318Q, or 318D; amino acids at amino acid position 340 are 340A, 340D, 340K, 340M, 340N, 340R, 340E, or 340P; amino acids at amino acid position 356 are 356D or 356E, and / or amino acids at amino acid position 406 are 406A, 406D, 406P, 406R, or 406K.

[0235] 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 132W or 132P at amino acid position 132; amino acid 175F at amino acid position 175; amino acid 188K or 188T at amino acid position 188; amino acid 281M at amino acid position 281; amino acid 294N, 294Q or 294K at amino acid position 294; amino acid 301K at amino acid position 301; amino acid 302F at amino acid position 302; amino acid 309D or 309E at amino acid position 309; amino acid 316Y at amino acid position 316; amino acid 318D at amino acid position 318; amino acid 340E or 340P at amino acid position 340; amino acid 356D at amino acid position 356; and / or 406K.

[0236] 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 278. 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: 278K, 278D, 278G, or 278E. 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: 278K, 278D, 278G, or 278E.

[0237] 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 278. 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: 278K, 278D, 278G, or 278E. 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: 278K, 278D, 278G, or 278E.

[0238] 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 271 and / or 315. 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 of 271K or 271L at amino acid position 271; and / or a substitution of 315T, 315E, or 315V at amino acid position 315. 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: an amino acid at amino acid position 271 being 271K or 271L; and / or an amino acid at amino acid position 315 being 315T, 315E, or 315V. In some embodiments, based on any one of the amino acids or amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may include the following substitutions relative to the amino acid sequence shown in SEQ ID NO:2:

[0239] 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 271 and / or 315. 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 of 271K at amino acid position 271; and / or a substitution of 315T, 315E, or 315V at amino acid position 315. 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: an amino acid of 271K at amino acid position 271; and / or an amino acid of 315T, 315E, or 315V at amino acid position 315. 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 the following substitutions relative to the amino acid sequence shown in SEQ ID NO:2:

[0240] A123N / D124N / N126L / H133Y / K136A / H140R / H142D / S148D / D152S / Y156H / Q178K / T181E / Y205D / N265G / S320A.

[0241] 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:2:

[0242] T116E / A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134M / K136A / H140R / H142D / S148D / D152S / Y156H / Q178K / T181E / Y205D / N265G / S320A;

[0243] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N26 5G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0244] T116K / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0245] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q17 8K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0246] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0247] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0248] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0249] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134D / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / E271K / N278D / H281M / A294K / G301K / Y302F / N309D / K315E / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0250] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278G / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0251] T116M / A123N / D124N / N126L / R127K / V128E / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294N / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0252] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0253] T116E / A123N / D124N / N126L / R127K / V128P / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0254] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0255] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294Q / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0256] T116E / A123N / D124N / N126L / R127K / V128A / E132P / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0257] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278E / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0258] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0259] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309E / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0260] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0261] T116E / A123N / D124N / N126L / R127E / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0262] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134D / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0263] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309E / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0264] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0265] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0266] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0267] T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0268] T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0269] T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0270] T116E / A123K / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / A294L / G301N / Y302W / L308F / N309A / H316A / L318E / S320A / Q340D / S356D / H406P;

[0271] T116Q / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316Y / L318D / S320A / Q340A / S356D / H406K;

[0272] T116M / A123N / N126C / R127K / V128E / E132P / R134K / Y156H / Y175W / T181E / G191C / Y205D / N265G / E271L / N278K / H281F / A294K / G301K / N309D / H316Y / L318D / S320A / Q340A / S356D / H406K;

[0273] T116E / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281V / A294L / G301N / Y302W / L308F / N309E / H316Q / L318E / S320A / Q340E / S356D / H406P;

[0274] T116E / A123N / N126C / R127H / V128E / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281M / A294Y / G301R / Y302V / L308F / N309E / H316A / L318E / S320A / Q340D / S356D / H406P;

[0275] T116E / A123K / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281M / A294V / G301V / Y302W / L308F / N309E / H316Q / L318E / S320A / Q340D / S356D / H406P;

[0276] T116E / A123Y / N126C / R127H / V128E / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281I / A294Q / G301K / Y302V / L308F / N309E / H316Y / L318E / S320A / S356D / H406P;

[0277] T116E / A123Q / N126C / R127K / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281M / G301K / Y302W / L308F / H316R / L318E / S320A / Q340E / S356D / H406P;

[0278] T116E / A123Y / N126C / R127K / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281V / A294Y / G301T / Y302A / L308F / N309E / L318E / S320A / Q340K / S356D / H406P;

[0279] T116E / A123Y / N126C / V128K / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281I / A294K / G301E / Y302L / L308F / N309D / L318E / S320A / Q340K / S356D / H406P;

[0280] T116E / A123Y / N126C / R127K / V128Q / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281M / A294L / G301R / Y302W / L308F / N309E / L318E / S320A / Q340A / S356D / H406P;

[0281] T116E / A123K / N126C / V128I / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281V / G301N / Y302V / L308F / N309E / H316R / L318E / S320A / Q340D / S356D / H406P;

[0282] T116E / A123E / N126C / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281I / A294T / G301K / Y302V / L308F / N309E / H316Q / L318E / S320A / Q340A / S356D / H406P;

[0283] T116E / A123N / N126C / V128T / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281V / A294L / G301L / Y302L / L308F / N309E / H316Q / L318E / S320A / Q340D / S356D / H406P;

[0284] T116E / A123R / N126C / R127K / V128I / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281I / A294E / G301K / Y302W / L308F / N309E / H316L / L318E / S320A / Q340A / S356D / H406P;

[0285] T116E / A123N / N126C / R127Q / V128A / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281F / G301N / Y302W / L308F / N309E / H316Y / L318E / S320A / Q340K / S356D / H406P;

[0286] T116E / A123H / N126C / V128E / E132P / R134K / Y156H / T181G / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281M / A294L / G301T / Y302G / L308F / N309E / H316A / L318E / S320A / Q340D / S356D / H406P;

[0287] T116E / A123N / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / A294D / G301N / Y302W / L308F / N309Q / H316A / L318E / S320A / Q340K / S356D / H406A;

[0288] T116E / A123E / N126C / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281I / A294L / Y302V / L308F / N309E / H316R / L318E / S320A / Q340A / S356D / H406P;

[0289] T116E / A123R / N126C / V128M / E132P / R134K / Y156H / T181N / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294S / G301A / Y302W / L308F / N309E / L318E / S320A / Q340D / S356D / H406A;

[0290] T116E / A123H / N126C / V128Q / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281C / A294V / G301T / Y302W / L308F / N309E / H316F / L318E / S320A / Q340D / S356D / H406P;

[0291] T116E / A123K / N126C / R127K / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281I / A294L / G301N / Y302V / L308F / N309E / H316R / L318E / S320A / Q340K / S356D / H406P;

[0292] T116M / A123N / N126C / R127K / V128E / E132P / R134S / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281F / A294K / G301Q / N309D / H316Y / L318D / S320A / Q340A / S356D / H406K;

[0293] T116E / A123N / N126C / R127K / V128E / E132V / R134K / Y156H / T181E / G191C / Y205D / N265G / E271L / N278K / H281F / G301K / N309D / H316Y / L318D / S320A / Q340A / S356D / H406K;

[0294] T116D / A123N / N126C / R127K / E132V / R134K / Y156H / Y175F / T181E / P188E / G191C / Y205D / N265G / E271L / N278K / H281F / A294K / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0295] T116N / A123N / N126C / R127K / V128D / E132V / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281F / G301T / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0296] T116M / A123N / N126C / R127K / V128D / E132P / R134E / Y156H / Y175F / P188T / G191C / Y205D / N265G / E271L / N278K / H281F / A294K / G301K / N309D / H316V / L318E / S320A / Q340A / S356D / H406P;

[0297] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281L / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0298] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0299] T116K / A123K / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340P / S356D / H406P;

[0300] T116D / A123R / N126C / R127K / V128E / E132V / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0301] T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0302] T116L / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0303] T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0304] T116K / A123E / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0305] T116L / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0306] T116K / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0307] T116E / A123E / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406K;

[0308] T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / H316V / L318E / S320A / Q340A / S356D / H406P;

[0309] T116E / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / N309E / H316V / L318D / S320A / Q340E / S356D / H406K;

[0310] T116E / A123K / N126C / R127K / V128E / E132P / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294L / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0311] T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0312] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0313] T116M / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0314] T116K / A123K / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281L / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0315] T116K / A123N / N126C / R127K / V128E / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0316] T116E / A123K / N126C / R127K / V128E / E132P / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318D / S320A / Q340E / S356D / H406P;

[0317] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294E / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0318] T116E / A123N / N126C / R127K / V128I / E132T / R134K / Y156H / T181N / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294N / G301Y / Y302F / L308F / N309D / H316Y / L318E / S320A / Q340K / S356D / H406A;

[0319] T116E / A123N / N126C / V128I / E132K / R134K / Y156H / T181S / P188S / G191C / Y205D / N265G / E271L / N278K / H281M / A294N / G301A / Y302F / L308F / N309E / H316Y / L318E / S320A / S356E / H406P;

[0320] T116E / A123I / N126C / V128M / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / A294Y / G301Q / L308F / N309E / H316M / L318E / S320A / Q340K / S356D / H406P;

[0321] T116E / A123K / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340E / S356D / H406P;

[0322] T116D / A123N / N126C / R127K / V128R / E132V / R134Q / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406K;

[0323] T116D / A123N / N126C / R127K / V128E / E132V / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0324] T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340A / S356D / H406P;

[0325] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0326] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / H316V / L318E / S320A / Q340A / S356D / H406P;

[0327] T116K / A123R / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0328] T116E / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406K;

[0329] T116E / A123K / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340A / S356D / H406P;

[0330] T116L / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318D / S320A / S356D / H406P;

[0331] T116D / A123N / N126C / R127K / V128E / E132V / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0332] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0333] T116D / A123K / N126C / R127K / V128E / E132V / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0334] T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0335] T116E / A123N / N126C / R127K / V128E / E132P / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406K;

[0336] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294L / G301K / Y302F / N309D / H316V / L318E / S320A / Q340E / S356D / H406P;

[0337] T116E / A123K / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0338] T116K / A123N / N126C / R127K / V128E / E132P / R134W / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340A / S356D / H406P;

[0339] T116M / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0340] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340E / S356D / H406P;

[0341] T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0342] T116L / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / H316V / L318D / S320A / Q340A / S356D / H406P;

[0343] T116K / A123E / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0344] T116K / A123N / N126C / R127K / V128E / E132P / R134W / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P;

[0345] T116K / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0346] T116K / A123N / N126C / R127K / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340A / S356D / H406P;

[0347] T116K / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406K;

[0348] T116E / A123K / N126C / V128E / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281M / A294E / G301R / Y302V / L308F / N309E / H316F / L318E / S320A / Q340M / S356D / H406P;

[0349] T116E / A123N / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / G301Y / Y302W / L308F / N309E / H316M / L318E / S320A / Q340R / S356D / H406P;

[0350] T116E / A123K / N126C / V128T / E132P / R134K / Y156H / T181A / P188S / G191C / Y205D / N265G / E271L / L274M / N278K / H281V / A294K / G301S / Y302F / L308F / N309E / H316M / L318E / S320A / Q340R / S356D / H406P;

[0351] I49Y / T116M / A123K / N126C / R127K / V128M / E132P / R134P / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0352] A39C / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / N265G / E271L / N278K / H281M / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P;

[0353] A39C / I49V / T116M / A123K / N126C / R127Q / V128E / E132P / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P;

[0354] T116M / A123K / N126C / R127Q / V128E / E132P / R134T / S148N / Y156H / Y175F / T181E / G191C / Y205N / I212L / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K;

[0355] A39C / I49Y / T116M / A123K / N126C / R127K / V128M / E132P / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0356] A39T / T116M / A123K / N126C / R127K / V128M / E132P / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0357] A39T / I49Y / T116M / A123K / N126C / R127Q / V128E / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294V / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K;

[0358] I49Y / T116M / A123K / N126C / R127Q / V128M / E132P / Y156H / Y175F / T181E / G191C / Y205N / I212L / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P / S417P;

[0359] A39C / I49Y / T116M / A123K / N126C / R127Q / V128M / E132P / R134T / Y156H / Y175F / T181E / G191C / Y205N / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K;

[0360] A39T / T116M / A123K / N126C / R127H / V128E / E132P / R134T / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P;

[0361] T116E / A123E / N126C / V128I / E132K / R134K / Y156H / T181D / P188N / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301T / Y302L / L308F / N309E / H316Q / L318E / S320A / Q340N / S356D / H406P;

[0362] T116E / A123T / N126C / R127H / V128I / E132P / R134K / Y156H / Y175F / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281V / A294Q / G301N / Y302F / L308F / N309D / H316F / L318E / S320A / Q340D / S356D / H406P;

[0363] T116E / A123K / N126C / R127K / V128M / E132K / R134T / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301S / Y302W / L308F / N309E / H316Q / L318E / S320A / Q340D / S356D / H406P;

[0364] T116E / A123N / N126C / R127E / V128T / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281I / A294Q / G301S / Y302F / L308F / N309D / H316E / L318E / S320A / Q340K / S356D / H406D;

[0365] T116E / A123T / N126C / V128E / E132T / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281I / A294R / G301N / Y302F / L308F / N309E / H316Y / L318Q / S320A / Q340A / S356D / H406A;

[0366] T116E / A123N / N126C / R127K / V128E / E132V / R134K / Y156H / T181S / P188N / G191C / Y205D / N265G / E271L / N278K / H281L / A294Q / G301V / Y302V / L308F / N309E / H316Q / L318Q / S320A / Q340D / S356E / H406P;

[0367] T116E / A123E / N126C / R127K / V128R / E132T / R134E / Y156H / T181S / P188E / G191C / Y205D / N265G / E271L / N278K / H281M / A294E / G301R / Y302A / L308F / N309E / V313L / H316Y / L318E / S320A / Q340R / S356D / H406A;

[0368] T116E / A123V / N126C / R127H / V128Q / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274M / N278K / H281T / A294E / G301R / Y302G / L308F / N309E / H316Y / L318E / S320A / Q340E / S356D / H406P;

[0369] T116E / A123N / N126C / V128T / E132P / R134V / Y156H / T181R / P188T / G191C / Y205D / N265G / E271L / N278K / H281I / A294E / G301R / Y302W / L308F / N309A / V313L / H316F / L318E / S320A / Q340E / S356D / H406P;

[0370] T116E / A123E / N126C / V128E / E132P / R134K / Y156H / T181N / P188S / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301N / Y302W / L308F / N309H / V313L / H316W / L318D / S320A / Q340D / S356D / H406P;

[0371] T116E / A123N / N126C / R127K / V128I / E132P / R134T / Y156H / T181N / P188N / G191C / Y205D / N265G / E271L / N278K / H281M / A294D / G301R / Y302A / L308F / L318E / S320A / Q340N / S356D / H406P;

[0372] T116E / A123Y / N126C / R127E / V128I / E132D / R134A / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281V / A294V / G301S / Y302F / L308F / N309E / H316F / L318E / S320A / Q340E / S356D / H406P;

[0373] T116E / A123E / N126C / V128I / E132T / R134T / Y156H / T181N / P188E / G191C / Y205D / N265G / E271L / L274I / N278K / H281M / A294D / G301N / Y302F / L308F / N309E / V313L / H316Q / L318E / S320A / Q340E / S356D / H406P;

[0374] K23M / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0375] L61M / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0376] K106L / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0377] T116M / A123N / N126C / R127K / V128E / E132P / R134M / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0378] T116M / A123N / N126C / R127K / V128E / E132P / K154Y / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0379] T116M / A123N / N126C / R127K / V128E / E132P / Y156F / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0380] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172E / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0381] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242L / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0382] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / K276S / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0383] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0384] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301H / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0385] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / K306H / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0386] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406P;

[0387] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / D407E;

[0388] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / R456L;

[0389] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242F / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K;

[0390] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406R;

[0391] W114M / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / K306Y / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0392] W114M / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301R / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0393] W114F / T116M / A123N / N126C / R127K / V128E / E132P / K154R / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0394] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172W / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302F / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0395] W114L / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / R456V;

[0396] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301R / Y302F / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0397] W114L / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294N / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0398] W114F / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / D407R;

[0399] T116M / A123N / N126C / R127K / V128E / E132P / K154L / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302W / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0400] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302W / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / R456L;

[0401] Y62W / T116M / A123N / N126C / R127K / V128E / E132P / R134F / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0402] Y62W / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / K306Y / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0403] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302W / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / D407R;

[0404] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172K / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / K306R / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0405] Y62W / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172L / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0406] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172K / Y175F / T181E / G191C / Y205D / R242F / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0407] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242T / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0408] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242S / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0409] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242A / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0410] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242S / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406P;

[0411] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242T / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406P;

[0412] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242N / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0413] T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242A / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406P;

[0414] K23S / T116M / A123N / N126C / R127K / V128E / E132P / R134E / K154T / W155Y / Y156H / Y175F / T181E / G191C / Y205D / R242E / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370Q / H406P;

[0415] K23Q / R74A / T116M / A123N / N126C / R127K / V128E / E132P / R134V / Y156H / Y175F / T181E / G191C / Y205D / R242E / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370H / H406P;

[0416] I49A / T116M / A123K / N126C / R127Q / V128M / E132P / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K;

[0417] T116M / A123N / N126C / R127H / V128E / E132P / R134T / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P;

[0418] A39T / I49Y / T116M / A123K / N126C / R127Q / V128I / E132P / Y156H / Y175F / T181E / G191C / Y205N / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K;

[0419] I49V / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P;

[0420] A39T / I49Y / T116M / A123K / N126C / R127K / V128I / E132P / R134T / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P;

[0421] A39T / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / Y205D / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K;

[0422] I49Y / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K;

[0423] I49Y / T116M / A123K / N126C / R127Q / V128E / E132P / R134T / S148N / Y156H / Y175F / T181E / G191C / Y205N / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K;

[0424] I49A / T116M / A123K / N126C / R127Q / V128E / E132P / R134T / S148N / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K;

[0425] T116M / A123K / N126C / R127Q / V128M / E132P / R134T / Y156H / Y175F / T181E / G191C / Y205N / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K;

[0426] A39C / I49Y / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0427] A39C / I49A / T116M / A123N / N126C / R127H / V128E / E132P / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P;

[0428] A39T / T116M / A123K / N126C / R127K / V128E / E132P / R134P / Y156H / Y175F / T181E / G191C / Y205N / I212L / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K;

[0429] A39T / T116M / A123K / N126C / R127Q / V128E / E132P / Y156H / Y175F / T181E / G191C / I212L / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P;

[0430] I49V / T116M / A123K / N126C / R127Q / V128M / E132P / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P / S417P;

[0431] I49Y / T116M / A123N / N126C / R127Q / V128E / E132P / R134T / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P;

[0432] A39C / I49V / T116M / A123K / N126C / R127Q / V128I / E132P / S148N / Y156H / Y175F / T181E / G191C / I212 L / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K / S417P;

[0433] A39T / I49Y / T116M / A123K / N126C / R127H / V128E / E132P / Y156H / Y175F / T181E / G191C / T213K / N 265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0434] I49Y / T116M / A123K / N126C / R127Q / V128E / E132P / S148N / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294V / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P;

[0435] A39C / I49V / T116M / A123K / N126C / R127K / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / T 213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P;

[0436] A39T / I49Y / T116M / A123K / N126C / R127H / V128E / E132P / S148N / Y156H / Y175F / T181E / G191C / Y205N / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P; and

[0437] A39C / I49Y / T116M / A123K / N126C / R127Q / V128E / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / N 265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K / S417P.

[0438] 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 126, 191, 271, and / or 278. 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 the group consisting of 126C, 191C, 271L, and / or 278K. 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 amino acids selected from the group consisting of 126C, 191C, 271L, and / or 278K.

[0439] 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 116, 123, 127, 132, 134, 175, 181, 281, 294, 301, 309, 315, 316, 318, 340, 356 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: substitutions at amino acid position 116 such as 116N, 116Q, 116M, 116D, 116E, 116K, 116L, or 116R; substitutions at amino acid position 123 such as 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123Y, 123N, 123F, 123L, or 123T; and substitutions at amino acid position 127 such as 127E, 127H, 127E, 127Q, 123Q, 123R, 123V, 123Y, 123N, 123F, 123L, or 123T. 27Q or 127K; substitutions at amino acid position 132 are: 132D, 132K, 132T, 132V, 132W, or 132P; substitutions at amino acid position 134 are: 134A, 134F, 134P, 134Q, 134S, 134V, 134M, 134K, 134E, 134W, or 134T; substitutions at amino acid position 175 are: 175F or 175W; substitutions at amino acid position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E; substitutions at amino acid position 281 are: 281C, 281F, ... Substitutions at amino acid position 294 include: 294D, 294E, 294L, 294N, 294R, 294S, 294T, 294V, 294Y, 294K, or 294Q; substitutions at amino acid position 301 include: 301A, 301E, 301H, 301L, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y, or 301K; substitutions at amino acid position 309 include: 309R, 309A, 309H, 309Q, 309E, or 309D; substitutions at amino acid position 315 include: The substitutions at amino acid position 316 are: 316A, 316E, 316F, 316L, 316M, 316Q, 316R, 316V, 316W, 316E, or 316Y; the substitutions at amino acid position 318 are: 318E, 318Q, or 318D; the substitutions at amino acid position 340 are: 340D, 340K, 340M, 340N, 340R, 340E, 340P, or 340A; the substitutions at amino acid position 356 are: 356D or 356E, and / or the substitutions at amino acid position 406 are: 406A, 406D, 406P, 406R, or 406K.

[0440] 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 116, 123, 127, 132, 134, 175, 181, 281, 294, 301, 309, 315, 316, 318, 340, 356 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 116 of 116M, 116D, 116E, 116K, 116L, or 116R; a substitution at amino acid position 123 of 123N, 123F, 123L, or 123T; a substitution at amino acid position 127 of 127K; a substitution at amino acid position 132 of 132W or 132P; a substitution at amino acid position 134 of 134M, 134K, 134E, 134W, or 134T; and a substitution at amino acid position 175 of 134M, 134K, 134E, 134W, or 134T. 175F; substitution at amino acid position 181: 181E; substitution at amino acid position 281: 281M; substitution at amino acid position 294: 294K or 294Q; substitution at amino acid position 301: 301K; substitution at amino acid position 309: 309E or 309D; substitution at amino acid position 315: 315V; substitution at amino acid position 316: 316Y; substitution at amino acid position 318: 318D; substitution at amino acid position 340: 340E, 340P or 340A; substitution at amino acid position 356: 356D or 356E, and / or substitution at amino acid position 406: 406K.

[0441] 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 acids at amino acid position 116 being: 116N, 116Q, 116M, 116D, 116E, 116K, 116L, or 116R; amino acids at amino acid position 123 being: 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123Y, 123N, 123F, 123L, or 123T; and amino acids at amino acid position 127 being: 127E, 127H, 127T, 127E, 127Q, 127L, 127T, 127E, 127Q ... 7Q or 127K; Amino acids at position 132 are: 132D, 132K, 132T, 132V, 132W, or 132P; Amino acids at position 134 are: 134A, 134F, 134P, 134Q, 134S, 134V, 134M, 134K, 134E, 134W, or 134T; Amino acids at position 175 are: 175F or 175W; Amino acids at position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E; Amino acids at position 281 are: 281C, 281F, ... 281I, 281L, 281T, 281V, or 281M; amino acids at amino acid position 294 are: 294D, 294E, 294L, 294N, 294R, 294S, 294T, 294V, 294Y, 294K, or 294Q; amino acids at amino acid position 301 are: 301A, 301E, 301H, 301L, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y, or 301K; amino acids at amino acid position 309 are: 309R, 309A, 309H, 309Q, 309E, or 309D; amino acids at amino acid position 315 are... : 315V; Amino acids at amino acid position 316 are: 316A, 316E, 316F, 316L, 316M, 316Q, 316R, 316V, 316W, 316E or 316Y; Amino acids at amino acid position 318 are: 318E, 318Q or 318D; Amino acids at amino acid position 340 are: 340D, 340K, 340M, 340N, 340R, 340E, 340P or 340A; Amino acids at amino acid position 356 are: 356D or 356E, and / or Amino acids at amino acid position 406 are: 406A, 406D, 406P, 406R or 406K.

[0442] 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 116 being 116M, 116D, 116E, 116K, 116L, or 116R; amino acid at amino acid position 123 being 123N, 123F, 123L, or 123T; amino acid at amino acid position 127 being 127K; amino acid at amino acid position 132 being 132W or 132P; amino acid at amino acid position 134 being 134M, 134K, 134E, 134W, or 134T; and amino acid at amino acid position 175 being 17... 5F; the amino acid at amino acid position 181 is: 181E; the amino acid at amino acid position 281 is: 281M; the amino acid at amino acid position 294 is: 294K or 294Q; the amino acid at amino acid position 301 is: 301K; the amino acid at amino acid position 309 is: 309E or 309D; the amino acid at amino acid position 315 is: 315V; the amino acid at amino acid position 316 is: 316Y; the amino acid at amino acid position 318 is: 318D; the amino acid at amino acid position 340 is: 340E, 340P or 340A; the amino acid at amino acid position 356 is: 356D or 356E, and / or the amino acid at amino acid position 406 is: 406K.

[0443] In some embodiments, in addition to 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 128, 188, and / or 302. In some embodiments, in addition to 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: substitutions at amino acid position 128 of 128I, 128K, 128M, 128Q, 128R, 128T, 128E, 128D, or 128A; substitutions at amino acid position 188 of 188E, 188G, 188N, 188S, 188K, 188T, 188D, or 188R; and / or substitutions at amino acid position 302 of 302A, 302G, 302L, 302V, 302W, or 302F. In some embodiments, for the modification method of amylases having a similar sequence to Bacillus licheniformis α-amylase or its mutants, based on any of the amino acid substitutions or combinations thereof described above, the resulting mutants may further include one or more amino acids selected from the group consisting of: amino acids at amino acid position 128 being 128I, 128K, 128M, 128Q, 128R, 128T, 128E, 128D or 128A; amino acids at amino acid position 188 being 188E, 188G, 188N, 188S, 188K, 188T, 188D or 188R; and / or amino acids at amino acid position 302 being 302A, 302G, 302L, 302V, 302W or 302F.

[0444] In some embodiments, in addition to 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 128, 188, and / or 302. In some embodiments, in addition to 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: substitutions at amino acid position 128 of 128E, 128D, or 128A; substitutions at amino acid position 188 of 188 of 188K, 188T, 188D, or 188R; and / or substitutions at amino acid position 302 of 302F. 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 128 being 128E, 128D or 128A; amino acid at amino acid position 188 being 188K, 188T, 188D or 188R; and / or amino acid at amino acid position 302 being 302F.

[0445] 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:2:

[0446] N265G / E271L / N126C / S320A / Y156H / Y205D / N278K / G191C; and

[0447] N265G / E271L / N126C / S320A / Y156H / Y205D / N278K / G191C / R127K / H406K / H281M / L318D / G301K / T181E / H316Y / Y175F / E132W / S356D / K315V / A294K.

[0448] 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:2:

[0449] N126C / G191C / S320A / Y156H / H205D / N265G;

[0450] T116D / A123L / N126C / R127K / V128E / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0451] T116K / A123N / N126C / R127K / E132W / R134W / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0452] T116M / A123N / N126C / R127K / V128D / E132W / R134M / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0453] T116E / A123N / N126C / R127K / V128E / E132P / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0454] T116E / A123N / N126C / R127K / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0455] T116K / A123F / N126C / R127K / V128E / E132W / R134W / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0456] T116L / A123N / N126C / R127K / V128E / E132W / R134W / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0457] T116E / A123N / N126C / R127K / V128E / E132W / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309D / K315V / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0458] T116D / A123N / N126C / R127K / E132W / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0459] T116M / A123N / N126C / R127K / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309D / K315V / H316Y / L318D / S320A / Q340P / S356D / H406K;

[0460] T116E / A123N / N126C / R127K / V128E / E132W / R134T / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309D / K315V / H316Y / L318D / S320A / Q340A / S356D / H406K;

[0461] T116E / A123N / N126C / R127K / V128D / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0462] T116K / A123N / N126C / R127K / V128E / E132W / R134W / Y156H / Y175F / T181E / P188D / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0463] T116R / A123N / N126C / R127K / V128D / E132W / R134E / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0464] T116K / A123L / N126C / R127K / V128E / E132W / R134W / Y156H / Y175F / T181E / P188R / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / S356D / H406K;

[0465] T116M / A123T / N126C / R127K / V128D / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356E / H406K;

[0466] T116D / A123F / N126C / R127K / V128E / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0467] T116M / A123N / N126C / R127K / V128E / E132W / R134M / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0468] T116D / A123N / N126C / R127K / V128D / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0469] T116M / A123N / N126C / R127K / V128A / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0470] T116M / A123F / N126C / R127K / V128D / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0471] T116M / A123N / N126C / R127K / V128A / E132W / R134E / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0472] T116M / A123F / N126C / R127K / V128A / E132W / R134E / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0473] T116M / A123N / N126C / R127K / V128A / E132W / R134M / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356E / H406K;

[0474] T116D / A123N / N126C / R127K / V128A / E132W / R134K / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0475] T116M / A123N / N126C / R127K / V128E / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0476] T116M / A123F / N126C / R127K / V128A / E132W / R134E / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K;

[0477] T116M / A123N / N126C / R127K / V128D / E132W / R134E / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; and

[0478] T116M / A123N / N126C / R127K / V128E / E132W / R134E / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K.

[0479] 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 123, 127, 131, 133, and / or 134. 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: substitutions at amino acid position 123 of 123N, 123Y, or 123F; substitutions at amino acid position 127 of 127K or 127E; substitutions at amino acid position 131 of 131E, 131D, or 131K; substitutions at amino acid position 133 of 133Y or 133F; and / or substitutions at amino acid position 134 of 134K, 134E, 134M, 134T, 134W, 134N, or 134D. 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 123 being 123N, 123Y, or 123F; amino acid at amino acid position 127 being 127K or 127E; amino acid at amino acid position 131 being 131E, 131D, or 131K; amino acid at amino acid position 133 being 133Y or 133F; and / or amino acid at amino acid position 134 being 134K, 134E, 134M, 134T, 134W, 134N, or 134D.

[0480] In some embodiments, in addition to any of the amino acid substitutions or combinations thereof described above, the α-amylase mutant of the present invention may further comprise substitutions at amino acid positions 116 and / or 132. In some embodiments, in addition to 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: substitutions at amino acid position 116 of 116E, 116K, 116M, 116R, 116D, or 116W; and / or substitutions at amino acid position 132 of 132W, 132P, 132A, or 132K. 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 acids at amino acid position 116 being 116E, 116K, 116M, 116R, 116D or 116W, and / or amino acids at amino acid position 132 being 132W, 132P, 132A or 132K.

[0481] 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 48, 49, 52, 68, 124, 128, 178, 181, 188, 271, 272, 275, 278, 279, 280, 281, 283, 291, 294, 298, 301, 307, 309, 312, 314, 315, 318, 319, 338, 372, 373, 374 and / or 375.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 48 of 48S; a substitution at amino acid position 49 of 49A; a substitution at amino acid position 52 of 52D; a substitution at amino acid position 68 of 68N; a substitution at amino acid position 124 of 124N; and substitutions at amino acid position 128 of 128A, 128D, 128E, 128I. 128K or 128L; substitution at amino acid position 178: 178E or 178K; substitution at amino acid position 181: 181E; substitution at amino acid position 188: 188K or 188T; substitution at amino acid position 271: 271K; substitution at amino acid position 272: 272R; substitution at amino acid position 275: 275E; substitution at amino acid position 278: 278D or 278E; substitution at amino acid position 279: 279Y; substitution at amino acid position 280: 2 80V; Substitution at amino acid position 281: 281M; Substitution at amino acid position 283: 283L; Substitution at amino acid position 291: 291K or 291R; Substitution at amino acid position 294: 294E; Substitution at amino acid position 298: 298A or 298S; Substitution at amino acid position 301: 301K; Substitution at amino acid position 307: 307I; Substitution at amino acid position 309: 309D; Substitution at amino acid position 312: 312L; Substitution at amino acid position 3 The substitution at amino acid position 14 is 314A; the substitution at amino acid position 315 is 315E; the substitution at amino acid position 319 is 319L; the substitution at amino acid position 338 is 338P, 338Q, or 338R; the substitution at amino acid position 372 is 372P or 372S; the substitution at amino acid position 373 is 373G, 373L, or 373T; the substitution at amino acid position 374 is 374D or 374S; and / or the substitution at amino acid position 375 is 375D, 375K, 375N, or 375T.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 48: 48S; amino acid at position 49: 49A; amino acid at position 52: 52D; amino acid at position 68: 68N; amino acid at position 124: 124N; amino acids at position 128: 128A, 128D, 128E, 128I ... 8K or 128L; Amino acid at position 178 is 178E or 178K; Amino acid at position 181 is 181E; Amino acid at position 188 is 188K or 188T; Amino acid at position 271 is 271K; Amino acid at position 272 is 272R; Amino acid at position 275 is 275E; Amino acid at position 278 is 278D or 278E; Amino acid at position 279 is 279Y; Amino acid at position 280 is 28 0V; The amino acid at amino acid position 281 is: 281M; The amino acid at amino acid position 283 is: 283L; The amino acid at amino acid position 291 is: 291K or 291R; The amino acid at amino acid position 294 is: 294E; The amino acid at amino acid position 298 is: 298A or 298S; The amino acid at amino acid position 301 is: 301K; The amino acid at amino acid position 307 is: 307I; The amino acid at amino acid position 309 is: 309D; The amino acid at amino acid position 312 is: 312L; The amino acid at amino acid position 3 The amino acid at position 14 is 314A; the amino acid at position 315 is 315E; the amino acid at position 319 is 319L; the amino acid at position 338 is 338P, 338Q, or 338R; the amino acid at position 372 is 372P or 372S; the amino acid at position 373 is 373G, 373L, or 373T; the amino acid at position 374 is 374D or 374S; and / or the amino acid at position 375 is 375D, 375K, 375N, or 375T.

[0482] 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:2:

[0483] N265G;

[0484] Y205D;

[0485] Y156H;

[0486] S320A;

[0487] Y156H / Y205D;

[0488] Y155H / N265G;

[0489] Y156H / S320A;

[0490] Y205D / N265G;

[0491] Y205D / S320A;

[0492] N265G / S320A;

[0493] 156H / 205D / 265G;

[0494] 156H / 205D / 320A;

[0495] 205D / 265G / 320A;

[0496] S320A / Y156H / H205D / N265G;

[0497] A48S / I49A / A52S / W68N / Y156H / Y205D / N265G / S320A;

[0498] A48S / I49A / A52D / W68N / Y156H / Y205D / N265G / S320A;

[0499] T116R / A123N / D124N / R127K / V128E / G131D / E132W / H133Y / R134M / Y156H / Y205D / N265G / S320A;

[0500] T116E / A123N / D124N / R127E / V128I / G131E / E132P / H133Y / R134M / Y156H / Y205D / N265G / S320A;

[0501] T116E / A123N / R127K / V128E / G131K / E132P / H133Y / R134T / Y156H / Y205D / N265G / S320A;

[0502] T116D / A123N / R127K / G131D / E132W / H133F / R134K / Y156H / Y205D / N265G / S320A;

[0503] T116E / A123N / R127K / V128I / G131D / E132A / H133F / R134M / Y156H / Y205D / N265G / S320A;

[0504] T116M / A123N / R127K / V128E / G131E / E132P / H133Y / R134M / Y156H / Y205D / N265G / S320A;

[0505] T116E / A123N / R127K / G131E / E132P / H133F / R134K / Y156H / Y205D / N265G / S320A;

[0506] T116K / A123N / R127K / V128I / G131D / E132W / H133Y / R134N / Y156H / Y205D / N265G / S320A;

[0507] T116W / A123N / R127K / V128E / E132W / H133Y / R134K / Y156H / Y205D / N265G / S320A;

[0508] T116M / A123N / D124N / R127K / V128I / G131D / H133Y / R134M / Y156H / Y205D / N265G / S320A;

[0509] T116E / A123N / R127K / V128E / G131E / E132W / H133Y / R134T / Y156H / Y205D / N265G / S320A;

[0510] T116E / A123N / G131D / E132W / H133Y / R134T / Y156H / Y205D / N265G / S320A;

[0511] T116E / A123N / D124N / R127K / V128I / G131D / E132W / H133Y / R134T / Y156H / Y205D / N265G / S320A;

[0512] T116E / A123N / D124N / R127K / G131D / E132W / H133Y / R134K / Y156H / Y205D / N265G / S320A;

[0513] T116R / A123N / D124N / R127K / V128E / G131D / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A;

[0514] T116R / A123N / R127K / G131D / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A;

[0515] T116K / A123N / D124N / R127K / V128L / G131D / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A;

[0516] T116K / A123N / R127K / V128I / G131D / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A;

[0517] T116K / A123Y / R127K / V128E / G131E / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A;

[0518] A123N / R127K / V128L / G131E / E132P / H133F / R134K / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0519] T116K / A123N / D124N / R127K / G131E / H133Y / R134W / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0520] T116K / A123N / R127K / V128E / G131E / E132P / H133F / R134W / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0521] T116K / A123N / R127K / V128I / G131D / E132W / H133Y / R134D / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0522] T116E / A123N / D124N / R127K / V128I / G131E / E132P / H133Y / R134W / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A;

[0523] T116R / A123N / R127K / G131D / E132W / H133Y / R134E / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0524] T116E / A123N / R127K / V128E / G131E / E132W / H133Y / R134K / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A;

[0525] T116E / A123N / R127K / V128I / G131K / E132P / H133F / R134K / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A;

[0526] A123N / R127K / V128I / G131E / H133Y / R134E / Y156H / Q178K / T181E / P188T / Y205D / N265G / S320A;

[0527] T116E / A123N / D124N / R127E / V128I / G131D / E132W / H133Y / R134N / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A;

[0528] T116D / A123N / R127K / G131E / E132K / H133Y / R134K / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0529] A123N / R127K / G131D / H133F / R134E / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A;

[0530] T116M / A123N / R127K / V128I / G131E / E132P / H133Y / R134M / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0531] A123N / D124N / R127K / V128I / G131E / E132P / H133Y / R134K / Y156H / Q178E / T181E / P188T / Y205D / N265G / S320A;

[0532] T116M / A123N / R127K / V128L / G131E / E132P / H133Y / R134K / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0533] T116K / A123F / R127K / V128D / G131E / E132P / H133Y / R134E / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0534] T116E / A123N / R127K / V128D / G131E / E132W / H133Y / R134K / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A;

[0535] T116K / A123Y / R127K / V128D / G131E / H133Y / R134E / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A;

[0536] T116E / A123N / D124N / R127K / V128L / G131K / E132P / H133Y / R134K / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A;

[0537] A123F / D124N / R127K / V128D / G131K / E132P / H133Y / R134K / Y156H / Q178E / T181E / P188T / Y205D / N265G / S320A;

[0538] T116K / A123N / R127K / V128L / G131D / E132A / H133Y / R134E / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0539] T116M / A123N / R127K / V128K / G131E / E132P / H133Y / R134M / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A;

[0540] T116E / A123Y / R127K / V128D / G131E / E132P / H133F / R134T / Y156H / Q178K / T181E / P188T / Y205D / N265G / S320A;

[0541] T116M / A123N / D124N / R127K / V128L / G131E / H133Y / R134E / Y156H / Q178K / T181E / P188T / Y205D / N265G / S320A;

[0542] Y156H / Y205D / N265G / E271K / N272R / N275E / N278E / F279Y / N280V / H281M / V283L / Q291K / A294E / Q298A / G301K / L307I / N309D / V312L / S314A / K315E / L318E / K319L / S320A;

[0543] Y156H / Y205D / N265G / N272R / N275E / N278D / F279Y / N280V / H281M / Q291R / A294E / Q298S / G301K / L307I / N309D / V312L / S314A / L318D / K319L / S320A;

[0544] Y156H / Y205D / N265G / S320A / D372P / S373T / Q374D / R375K;

[0545] Y156H / Y205D / N265G / S320A / T338Q / D372P / S373G / Q374D / R375K;

[0546] Y156H / Y205D / N265G / S320A / D372P / S373L / Q374S / R375N;

[0547] Y156H / Y205D / N265G / S320A / D372S / S373G / Q374S / R375D;

[0548] Y156H / Y205D / N265G / S320A / D372P / S373G / Q374S / R375K;

[0549] Y156H / Y205D / N265G / S320A / D372P / S373L / Q374D / R375T;

[0550] Y156H / Y205D / N265G / S320A / T338R / D372P / S373T / Q374S / R375D;

[0551] Y156H / Y205D / N265G / S320A / T338P / D372P / S373T / Q374D / R375N; and

[0552] Y156H / Y205D / N265G / S320A / D372P / S373T / Q374S / R375N.

[0553] In some embodiments, the α-amylase mutant of the present invention may contain substitutions at one or more of the following amino acid positions: 123, 127, 128, 134, 136, 142, 148, 178 and / or 181. 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 123 of 123N; a substitution at amino acid position 127 of 127K; a substitution at amino acid position 128 of 128E or 128I; a substitution at amino acid position 134 of 134E, 134K or 134M; a substitution at amino acid position 136 of 136A, 136D or 136E; a substitution at amino acid position 142 of 142D; a substitution at amino acid position 148 of 148T or 148D; a substitution at amino acid position 178 of 178K or 178E; and / or a substitution at amino acid position 181 of 181E. 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 123N at amino acid position 123; amino acid 127K at amino acid position 127; amino acid 128E or 128I at amino acid position 128; amino acid 134E, 134K, or 134M at amino acid position 134; amino acid 136A, 136D, or 136E at amino acid position 136; amino acid 142D at amino acid position 142; amino acid 148T or 148D at amino acid position 148; amino acid 178K or 178E at amino acid position 178; and / or amino acid 181E at amino acid position 181. 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 116. 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 the following substitutions: 116M, 116D, 116E, or 116K.

[0554] 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 101, 111, 114, 118, 124, 126, 131, 132, 133, 140, 152, 156, 167, 168, 169, 170, 171, 172 and / or 173. 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 101 of 101L; a substitution at amino acid position 111 of 111S; a substitution at amino acid position 114 of 114E or 114T; a substitution at amino acid position 118 of 118Q; a substitution at amino acid position 124 of 124N; a substitution at amino acid position 126 of 126L; a substitution at amino acid position 131 of 131D or 131E; and a substitution at amino acid position 132 of 1... 32P or 132W; substitution at amino acid position 133: 133Y; substitution at amino acid position 140: 140K or 140R; substitution at amino acid position 152: 152S; substitution at amino acid position 156: 156T; substitution at amino acid position 167: 167Y; substitution at amino acid position 168: 168K; substitution at amino acid position 169: 169T; substitution at amino acid position 170: 170N; substitution at amino acid position 171: 171K; substitution at amino acid position 172: 172K; and / or substitution at amino acid position 173: 173A.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 101: 101L; amino acid at amino acid position 111: 111S; amino acid at amino acid position 114: 114E or 114T; amino acid at amino acid position 118: 118Q; amino acid at amino acid position 124: 124N; amino acid at amino acid position 126: 126L; amino acid at amino acid position 131: 131D or 131E; amino acid at amino acid position 132: 13... 2P or 132W; the amino acid at amino acid position 133 is 133Y; the amino acid at amino acid position 140 is 140K or 140R; the amino acid at amino acid position 152 is 152S; the amino acid at amino acid position 156 is 156T; the amino acid at amino acid position 167 is 167Y; the amino acid at amino acid position 168 is 168K; the amino acid at amino acid position 169 is 169T; the amino acid at amino acid position 170 is 170N; the amino acid at amino acid position 171 is 171K; the amino acid at amino acid position 172 is 172K; and / or the amino acid at amino acid position 173 is 173A. 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 126, 191, 271 and / or 278. 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 126C, 191C, 271L, and / or 278K.

[0555] 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:2:

[0556] A123N / T181E / H142D / R127K;

[0557] A123N / T181E / H142D / R127K / K136N; and

[0558] A123N / T181E / H142D / R127K / K136N / Q178K.

[0559] 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:2:

[0560] A123N / N126L / R127K / V128E / E132P / H133Y / R134K / K136A / H140K / H142D / S148T / Y156H / Q178E / T181E / Y205D / N265G / S320A;

[0561] A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134K / K136A / H140R / H142D / S148T / Y156H / Q178E / T181E / Y205D / N265G / S320A;

[0562] A123N / D124N / N126L / R127K / V128E / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156T / Q178K / T181E;

[0563] T116E / A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134M / K136A / H140R / H142D / S148D / D152S / Y156T / Q178K / T181E;

[0564] A123N / N126L / R127K / V128E / E132P / H133Y / R134K / K136A / H140K / H142D / S148T / Y156T / Q178E / T181E;

[0565] A123N / N126L / R127K / V128E / H133Y / R134E / K136A / H140R / H142D / S148D / Y156T / Q178K / T181E;

[0566] A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134K / K136A / H140R / H142D / S148T / Y156T / Q178E / T181E;

[0567] T116D / A123N / N126L / R127K / V128E / E132W / H133Y / R134K / K136A / H140K / H142D / S148D / D152S / Y156T / Q178K / T181E;

[0568] V101L / A111S / T116M / A123N / R127K / V128I / G131E / R134E / K136A / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178K / T181E;

[0569] V101L / A111S / T116M / V118Q / A123N / R127K / V128I / G131E / R134E / K136A / H14 2D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178K / T181E;

[0570] V101L / A111S / T116K / A123N / R127K / V128I / G131E / R134E / K136A / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178E / T181E;

[0571] V101L / A111S / W114T / T116M / A123N / R127K / V128I / G131D / R134E / K136E / H14 2D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178K / T181E;

[0572] V101L / A111S / W114E / T116M / A123N / R127K / V128I / G131D / R134M / K136D / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178K / T181E; and

[0573] V101L / A111S / T116M / V118Q / A123N / R127K / V128I / G131E / R134E / K136A / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R173A / Q178K / T181E.

[0574] 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 comprise one or more amino acids selected from the group consisting of: 3(AAPF), 68W, 114W, 134R, 172R, 187D, 188P, 201Y, 205Y, 213T, 247Y, 360C, 416V, and 437W. In some embodiments, any parental or α-amylase mutant of the present invention may further comprise the following amino acids or the following substitutions: 3(AAPF) / 68W / 114W / 134R / 172R / 187D / 188P / 201Y / 205Y / 213T / 247Y / 360C / 416V / 437W. In some embodiments, any parent or α-amylase mutant of the present invention may further comprise the following substitutions: V3(AAPF) / H68W / D114W / L134R / N172R / S187D / N188P / F201Y / H205Y / K213T / H247Y / Q360C / D416V / R437W.

[0575] 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 has increased α-amylase activity and / or increased 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 90°C, or at least about 100°C, for example, 80°C-120°C, preferably 95°C-115°C, more preferably 100°C-110°C. "Low pH" refers to a pH value of 4.0-5.0, preferably about 4.5 or about 4.2.

[0576] 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%. In some embodiments, the parental α-amylase or other α-amylase mutant is mutant LE399 (SEQ ID NO:15) or mutant M35 (SEQ ID NO:2). In some embodiments, the parental α-amylase or other α-amylase mutant is any of the amylases shown in SEQ ID NO:1 or SEQ ID NO:3-14. 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 with 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 at 100°C and pH 4.5. In some embodiments, the enzyme activity assay is performed at 100°C and pH 4.2.

[0577] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention under high temperature conditions, particularly 80°C-120°C (e.g., about 100°C), 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 100°C 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 held at 100°C for 30 minutes is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the parental α-amylase or other α-amylase mutant is mutant LE399 or mutant M35. In some embodiments, the parental α-amylase or other α-amylase mutant is any of the amylases shown in SEQ ID NO:1 or SEQ ID NO:3-14. In some implementations, the enzyme activity of untreated mutants is measured at 70°C.

[0578] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C for 30 minutes to the residual enzyme activity % of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same treatment is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, or greater than 3. 30, greater than 3.40, greater than 3.50, greater than 3.60, greater than 3.70, greater than 3.80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, about 1.05, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00.

[0579] 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.

[0580] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention under low pH conditions, particularly pH 4.0-5.0 (e.g., about 4.5 or about 4.2), is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.5 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.2 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.2 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant at pH 4.5. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at pH 4.5 for 30 minutes is higher than that of the parental α-amylase 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 4.2 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 residual enzyme activity % of the α-amylase mutant of the present invention after being kept at pH 4.2 for 30 minutes is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutant after being kept at pH 4.5 for 30 minutes. In some embodiments, the parental α-amylase or other α-amylase mutant used for comparison with the mutant described herein is mutant LE399 or mutant M35. In some embodiments, the parental α-amylase or other α-amylase mutant is any one of the amylases shown in SEQ ID NO:1 or SEQ ID NO:3-14. In some implementations, the enzyme activity of untreated mutants is measured at pH 6.0.

[0581] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.5 or pH 4.2 to the residual enzyme activity % of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same treatment is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, and greater than 3. 50, greater than 3.60, greater than 3.70, greater than 3.80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, about 1.05, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00.

[0582] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.2 to the residual enzyme activity % of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant at pH 4.5 is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, greater than 3.50, greater than 3.60, greater than 3.70, greater than 3. .80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, about 1.05, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00.

[0583] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.5 is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at pH 6.0.

[0584] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at pH 4.2 is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at pH 6.0.

[0585] In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention under high temperature and low pH conditions, particularly 80°C-120°C and pH 4.0-5.0 (e.g., 100°C, pH 4.5 or 100°C, pH 4.2), is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention at 100°C and pH 4.5 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity of the α-amylase mutant of the present invention at 100°C and pH 4.2 is higher than that of the parental α-amylase or wild-type α-amylase or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention at 100°C and pH 4.2 is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutants at 100°C and pH 4.5. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.5 for 30 minutes is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.2 for 30 minutes is higher than that of the parental α-amylase, wild-type α-amylase, or other α-amylase mutants under the same conditions. In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.2 for 30 minutes is higher than that of the residual enzyme activity % of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant after being kept at 100°C and pH 4.5 for 30 minutes. In some embodiments, the parental α-amylase or other α-amylase mutant is mutant LE399 or mutant M35. In some embodiments, the parental α-amylase or other α-amylase mutant is any of the amylases shown in SEQ ID NO:1 or SEQ ID NO:3-14. In some embodiments, the enzyme activity assay of the untreated mutant is performed at 70°C and pH 6.0.

[0586] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C, pH 4.5 or 100°C, pH 4.2 for 30 minutes to the residual enzyme activity % of the parental α-amylase or wild-type α-amylase or other α-amylase mutant under the same treatment is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, and greater than 1.10, greater than 1.20, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, greater than 1.20, greater than 1.20, greater than 3.30, greater than 3.40, greater than 1.20, greater than 1.20, greater than 1.20, greater than 1.20, greater than 1.20, greater than 1.40, greater than 1.20, greater Values ​​of 3.50, greater than 3.60, greater than 3.70, greater than 3.80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, approximately 1.05, approximately 1.10, approximately 1.20, approximately 1.30, approximately 1.40, approximately 1.50, approximately 1.60, approximately 1.70, approximately 1.80, approximately 1.90, approximately 2.00, approximately 2.10, approximately 2.20, approximately 2.30, approximately 2.40, approximately 2.50, approximately 2.60, approximately 2.70, approximately 2.80, approximately 2.90, approximately 3.00, approximately 3.10, approximately 3.20, approximately 3.30, approximately 3.40, approximately 3.50, approximately 3.60, approximately 3.70, approximately 3.80, approximately 3.90, and approximately 4.00.

[0587] In some embodiments, the ratio of the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.2 for 30 minutes to the residual enzyme activity % of the parental α-amylase, wild-type α-amylase, or other α-amylase mutant after being kept at 100°C and pH 4.5 for 30 minutes is greater than 1.00, greater than 1.10, greater than 1.20, greater than 1.30, greater than 1.40, greater than 1.50, greater than 1.60, greater than 1.70, greater than 1.80, greater than 1.90, greater than 2.00, greater than 2.10, greater than 2.20, greater than 2.30, greater than 2.40, greater than 2.50, greater than 2.60, greater than 2.70, greater than 2.80, greater than 2.90, greater than 3.00, greater than 3.10, greater than 3.20, greater than 3.30, greater than 3.40, greater than 3.50, greater than 3.60, greater than 3.70. Greater than 3.80, greater than 3.90, greater than 4.0 or higher, such as, but not limited to, about 1.05, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00.

[0588] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.5 for 30 minutes is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at 70°C and pH 6.0.

[0589] In some embodiments, the residual enzyme activity % of the α-amylase mutant of the present invention after being kept at 100°C and pH 4.2 for 30 minutes is at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, for example, but not limited to, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the enzyme activity assay of the mutant without the corresponding treatment is performed at 70°C and pH 6.0.

[0590] This invention also relates to methods for improving the enzymatic activity, thermal stability, and / or low pH stability of α-amylase, including performing the above-described mutations on parental α-amylases (e.g., but not limited to α-amylase mutant M35). The above-described mutations can be performed using any method known in the art, such as site-directed mutagenesis, homologous recombination, etc.

[0591] 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 coding sequence for a signal peptide, enabling the mutant to be secreted extracellularly after expression.

[0592] The expression vector comprises a polynucleotide encoding the 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. Constitutive or inducible promoters can be used. Control sequences suitable for different host cells are well known to those skilled in the art. In some embodiments, the promoter may be the P43 gene promoter of Bacillus subtilis strain 168, the terminator may be the terminator of the amyQ α-amylase gene of Bacillus amyloliquefaciens, and the signal peptide sequence may be the sequence encoding the signal peptide of the Bacillus licheniformis amylase gene. In some embodiments, the promoter sequence may be the nucleotide sequence shown in SEQ ID NO:16. In some embodiments, the terminator sequence may be the nucleotide sequence shown in SEQ ID NO:17.

[0593] 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 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, a polynucleotide encoding an α-amylase mutant of the present invention is inserted into the plasmid vector pUC57 to prepare an expression vector for expressing the α-amylase mutant.

[0594] This invention also relates to recombinant host cells for expressing the α-amylase mutant of the present 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 a fungus from the genus *Aspergillus*, such as *Aspergillus niger*, *Aspergillus oryzae*, etc. In some embodiments, the host cell can be a bacterium, such as a bacterium from the genus *Bacillus*, such as *Bacillus subtilis*, *Bacillus licheniformis*, *Bacillus megaterium*, etc. In some embodiments, *Bacillus subtilis* is used as the host cell. In some embodiments, *Bacillus licheniformis* is used as the host cell. Methods for introducing polynucleotides into host cells are well known to those skilled in the art, such as chemical transformation (e.g., CaCl2 treatment), electroporation, gene guns, etc.

[0595] 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.

[0596] 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 subtilis to obtain host cells expressing the α-amylase mutant; (c) culturing the host cells to express the α-amylase mutant; and (d) recovering the α-amylase mutant.

[0597] 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.

[0598] 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.

[0599] 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.

[0600] 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.

[0601] Those skilled in the art will understand that in industrial production, starch liquefaction is typically carried out at high temperatures (e.g., 80°C-120°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.

[0602] 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.

[0603] 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.

[0604] 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.

[0605] Example 1: Construction of integration plasmids for M35 and its mutants

[0606] The gene sequence of M35 (SEQ ID NO:2) was synthesized by Nanjing Genscript Biotech Co., Ltd. after codon optimization. The M35 gene and its mutants were expressed using the amyL promoter (SEQ ID NO:16) of Bacillus licheniformis ATCC 9789 strain. The terminator was selected from the terminator sequence of the amyQ gene of Bacillus amyloliquefaciens α-amylase (SEQ ID NO:17), and the signal peptide was the Bacillus licheniformis amylase signal peptide (SEQ ID NO:18). The DNA fragment containing the promoter, signal peptide, complete M35 gene, and amyQ terminator was synthesized by Genscript. The M35 Bacillus licheniformis integrative plasmid pBSJ-M35-amyE was constructed using the following method:

[0607] Using genomic DNA from *Bacillus licheniformis* ATCC9789 as a template, the 800 bp upstream homologous arm of amyL (fragment 1) was amplified using primers amyL-5'-F and amyL-5'-R; the 734 bp downstream homologous arm of amyL (fragment 2) was amplified using primers amyL-3'-F and amyL-3'-R. Using the M35 gene synthesis plasmid as a template, a 1904 bp fragment (fragment 3) containing the promoter, signal peptide, M35, and amyQ terminator was amplified using primers M35-F and M35-R. These three fragments were then recombined using overlap PCR to obtain the complete knock-in fragment M35-amyE. The plasmid pBSJ was linearized with KpnI and XhoI, and then combined with the knock-in fragment M35-amyE using GenBuilder. TMHomologous recombination was performed using the Plus Cloning Kit (Genscript, catalog number: L00744) to construct the integrative plasmid pBSJ-M35-amyE. The M35 mutant integrative plasmid was generated by point mutation based on the pBSJ-M35-amyE integrative plasmid. The integrative plasmid sequence is shown in SEQ ID NO:19, and the primer sequences used are shown in Table 1. Underlined sequences indicate restriction enzyme sites.

[0608] Table 1 Primer names and sequences

[0609] Following the method described above for constructing integration plasmids for the M35 mutant, integration plasmids for each mutant, as shown in Table 2 below, were constructed. The difference is that the M35 gene was replaced with the gene for each mutant shown in Table 2.

[0610] Example 2: Construction of M35 and its mutant strains

[0611] Plasmid electroporation and verification: The pBSJ-M35-amyE integrative plasmid or its mutant integrative plasmid constructed in Example 1 was transformed into Bacillus licheniformis ATCC 9789 by electroporation. After incubation at 30°C and 220 rpm for 2-3 hours, the bacterial cells were plated onto LB agar plates containing 5 μg / ml erythromycin (formulation: 1% peptone, 0.5% yeast extract, 1% NaCl, 1.5% agar). After incubation at 30°C for 2-3 days, colony PCR was performed using primers M35-F and M35-R. The PCR product of the positive clone was 1749 bp.

[0612] Integration and validation of the target gene: The above positive clones were inoculated into LB liquid medium containing 0.5 μg / ml erythromycin and cultured overnight at 37°C and 220 rpm. The culture was then plated onto LB plates with a final concentration of 0.5 μg / ml and cultured overnight at 37°C. Colony PCR validation was performed using AmyL-test-F and M35-R primers. The PCR product of the positive clones was 2641 bp.

[0613] Elimination and verification of the erythromycin resistance gene: Integrative positive clones were inoculated into antibiotic-free LB broth and cultured overnight at 30°C and 220 rpm. Approximately 30 clones were simultaneously spotted onto both antibiotic-free LB broth and LB broth containing 0.5 μg / ml erythromycin. After overnight culture at 37°C, clones that grew on antibiotic-free LB broth but not on erythromycin-containing LB broth were selected. Colony PCR verification was performed using amyL-test-F and amyL-test-R. The PCR product of positive clones was 3462 bp. The PCR products of the positive clones were sequenced; clones with correct sequencing were identified as integrative positive clones.

[0614] The above methods were used to obtain Bacillus licheniformis expression strains LE399, M35 and the mutants shown in Table 2. The mutation sites of each mutant relative to the parent SEQ ID NO:2 are shown in Table 2.

[0615] Table 2 Mutant sites

[0616] Example 3: Shake-flask fermentation

[0617] The M35 strain and the mutant strain obtained in Example 2 were subjected to shake-flask fermentation. The experimental method is as follows:

[0618] Select appropriate amounts of bacterial cells and inoculate them into 20 ml of LB liquid seed medium (formula: 4.0% maltose syrup, 2.0% peptone, 0.1% yeast extract, 0.6% KH2PO4), and incubate overnight at 37℃ and 220 rpm. Then, inoculate 1% of the culture into 30 ml of fermentation medium (formula: 12.0% maltose syrup, 1.0% peptone, 1% yeast extract, 0.2% KH2PO4, 0.003% MnCl2), and incubate at 37℃ and 220 rpm for 48 h. Collect the fermentation broth and test the amylase activity.

[0619] Example 4: Detection of amylase activity

[0620] The definition of an amylase activity unit is: 1 mL of liquid enzyme liquefies 1 mg of soluble starch in 1 min at pH 6.0 and 70℃, which is 1 unit of enzyme activity, expressed as U / mL.

[0621] The percentage of heat-resistant residue refers to the percentage of enzyme activity after incubation at pH 4.5 or pH 4.2 and 100℃ for 30 minutes, relative to the enzyme activity before incubation (pH 6.0 and 70℃).

[0622] The enzyme activity detection 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 accurately for 5 min, 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. Quickly measure its absorbance value at a wavelength of 660 nm. According to Appendix B of GBT 24401-2009 "National Food Safety Standard for Food Additives and Enzyme Preparations for Food Industry", the concentration of the test enzyme solution is obtained. Then, according to the calculation formula X = C * N * 16.67 (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), the enzyme activity of the test sample is obtained.

[0623] Relative enzyme activity % refers to the enzyme activity of each amylase mutant relative to the parent, that is, the enzyme activity of the parent is set to 100%, and the enzyme activity of each mutant is relative to the enzyme activity of the parent.

[0624] When M35 (SEQ ID NO:2) was used as the parent, the experimental results of the relative enzyme activity % and the heat resistance residue % (at 100℃, pH 4.5 or pH 4.2) of each mutant are shown in Table 3.

[0625] Table 3. Relative enzyme activity (%) and residual heat resistance (%) of each mutant when M35 is the parent.

[0626] Example 5: Thermostable residual % of α-amylase mutant at 100°C, pH 4.5 / pH 4.2

[0627] The mutant expression strains shown in Table 4 below were constructed using the methods described in Examples 1-2 (where Bacillus subtilis ATCC 6051a was used instead of Bacillus licheniformis for the expression strains, and the mutations shown in Table 4 are the mutations of each mutant relative to the parent M35 (SEQ ID NO:2)). The mutants shown in Table 4 below were obtained using the method described in Example 3, and the relative enzyme activity % and thermostable residual enzyme activity % were detected using M35 as the parent at 100℃ and pH 4.5 using the method described in Example 4. The results are shown below:

[0628] Table 4. Relative enzyme activity (%) and residual heat resistance (%) of each mutant when M35 is the parent.

[0629] Example 6: Effect of reverse mutation on the percentage of thermostable residue in α-amylase mutants

[0630] Select mutants X136, X148, X155, and X158 shown in Table 5, and construct reversion mutants (i.e., no substitution relative to parent M35 at amino acid positions 123, 127, 128, 134, and 181) using the methods described in Examples 1-3. These mutants were named X136-1, X148-1, X155-1, and X158-1, respectively. The mutation sites of each mutant and reversion mutant relative to parent SEQ ID NO:2 are shown in Table 5.

[0631] Table 5. Mutants and Reversion Mutant Sites

[0632] The enzyme activity heat resistance residual percentage of the above mutant after being kept at 100°C and pH 4.2 for 30 minutes was detected using the method described in Example 4. Table 6 shows that the heat resistance residual percentage of the reversion mutant is lower than that of the mutant before the reversion mutation, which means that the substitution of the above site is beneficial to improving the stability of the enzyme at high temperature and / or low pH.

[0633] Table 6. Residual heat resistance (%) of each mutant and revertant mutant when M35 is the parent.

[0634] The amino acid and nucleotide sequences involved in this invention (partial):

[0635] SEQ ID NO:1 (Bacillus licheniformis BLA amino acid sequence)

[0636] SEQ ID NO:2 (M35 amino acid sequence)

[0637] SEQ ID NO:3

[0638] SEQ ID NO:4

[0639] SEQ ID NO:5

[0640] SEQ ID NO:6

[0641] SEQ ID NO:7

[0642] SEQ ID NO:8

[0643] SEQ ID NO:9

[0644] SEQ ID NO:10

[0645] SEQ ID NO:11

[0646] SEQ ID NO:12

[0647] SEQ ID NO:13

[0648] SEQ ID NO:14

[0649] SEQ ID NO:15 (LE399 amino acid sequence)

[0650] SEQ ID NO:16 (amyL promoter of Bacillus licheniformis ATCC 9789 strain)

[0651] SEQ ID NO:17 (amyQ terminator sequence of Bacillus amyloliquefaciens α-amylase gene)

[0652] SEQ ID NO:18 (Amino acid sequence of Bacillus licheniformis amylase signal peptide)

[0653] SEQ ID NO:19 (integration plasmid pBSJ-M35-amyE sequence)

[0654] 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

α-Amylase mutants containing amino acid positions 48, 49, 52, 68, 101, 111, 114, 116, 118, 123, 124, 126, 127, 128, 131, 132, 133, 134, 136, 140, 142, 148, 152, 156, 167, 168, 169, 170, 171, 172, 173, 175, 178, 181, 188, 191, 205. Insertion, deletion, and / or substitution at one or more of positions 265, 271, 272, 275, 278, 279, 280, 281, 283, 291, 294, 298, 301, 302, 307, 309, 312, 314, 315, 316, 318, 319, 320, 338, 340, 356, 372, 373, 374, 375, and 406, wherein the amino acid positions correspond to the positions in SEQ ID NO:1, wherein the mutant has at least 60% and less than 100% sequence identity with any of the amino acid sequences shown in SEQ ID NO:1-15 or their mature polypeptides, and wherein the mutant has α-amylase activity. The mutant of claim 1, comprising substitutions at one or more of the following positions: The amino acid at position 48 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S; The amino acid at position 49 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A, V or Y. The amino acid at position 52 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or S; The amino acid at position 68 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N; The amino acid at position 101 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L; The amino acid at position 111 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S; The amino acid at position 114 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by F, L, M, E or T. The amino acid position 116 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by N or Q or D or E or K or L or M or R or W. The amino acid at position 118 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably by Q; The amino acid positions 1, 2, and 3 are 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, H, I, K, Q, R, V, F, L, N, T, or Y. The amino acid at position 124 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N; The amino acid at position 126 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C or L. The amino acid at position 127 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably by H, Q, E, R, D, M, K, W, LE, or K. 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 by M or Q or R or T or A or D or E or I or K or L or P. The amino acid at position 131 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D, E or K. The amino acid position 132 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or T or V or A or K or P or W. The amino acid at position 133 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F or Y. The amino acid position 134 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or F or D or E or K or M or N or P or T or W. The amino acid at position 136 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A, D or E. The amino acid at position 140 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or R. At amino acid position 142, A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V are used, preferably D is used instead; The amino acid at position 148 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D, T or N. The amino acid at position 152 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably S; The amino acid at position 156 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably H or T or F. The amino acid at position 167 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y. The amino acid at position 168 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K; The amino acid at position 169 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably T; The amino acid at position 170 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably N; The amino acid at position 171 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K; The amino acid at position 172 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by E, K or L or W. The amino acid at position 173 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A; The amino acid at position 175 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably F or W. The amino acid at position 178 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or K. The amino acid at position 181 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by T or A or D or E or G or N or R or S. The amino acid at position 188 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by E or G or N or S or D or K or R or T. The amino acid at position 191 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C; The amino acid at position 205 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or N; The amino acid at position 265 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably G; The amino acid at position 271 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or L. The amino acid at position 272 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R; The amino acid at position 275 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E; The amino acid at position 278 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E or G or K. The amino acid at position 279 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, preferably Y. The amino acid at position 280 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably V; The amino acid at position 281 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably C or F or I or L or T or V or M. The amino acid at position 283 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L; The amino acid at position 291 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably K or R. 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 by D or E or K or L or N or Q or R or S or T or V or Y. The amino acid at position 298 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or S. The amino acid position 301 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or E or H or L or N or Q or R or S or T or V or Y or K. 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 A or G or L or V or W or F. The amino acid at position 307 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L or I. The amino acid at position 309 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by R or A or H or Q or D or E. The amino acid at position 312 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L; The amino acid at position 314 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A; The amino acid at position 315 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably E or T or V. The amino acid position 316 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or E or F or L or M or Q or R or V or W or E or Y. 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 Q, D or E; The amino acid at position 319 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably L; The amino acid at position 320 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A; The amino acid at position 338 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably P, Q or R. The amino acid at position 340 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or K or M or N or R or A or E or P. The amino acid at position 356 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or E; The amino acid at position 372 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably P or S. The amino acid at position 373 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by G, L or T. The amino acid at position 374 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or S. The amino acid at position 375 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably by D or K or N or T. The amino acid at position 406 is replaced by A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, preferably A or D or P or R or K. The amino acid position thereon corresponds to the position in SEQ ID NO:

1. The mutant as described in claim 1 or 2, comprising one or more substitutions selected from the group consisting of: A48S, I49A, I49V, I49Y, A52D, A52S, W68N, V101L, A111S, W114E, W114T, T116N, T116Q, T116D, T116E, T116K, T116L, T116M, T116R, T116W, V118Q, A123E, A123H, A123I, A123K, A123Q, A123R, A123V, A123F, A123L, A123N, A123T, A123Y, D124N, N126C, N126L, R127E. R127K, V128M, V128Q, V128R, V128T, V128A, V128D, V128E, V128I, V128K, V1 28L, V128P, G131D, G131E, G131K, E132D, E132T, E132V, E132A, E132K, E132P , E132W, H133F, H133Y, R134A, R134F, R134Q, R134S, R134V, R134D, R134E, R 134K, R134M, R134N, R134P, R134T, R134W, K136A, K136D, K136E, H140K, H140 R, H142D, S148D, S148T, S148N, D152S, Y156H, Y156T, Y156F, E167Y, S168K, R169T, K170N, L171K, R172E, R172L, R172W, R172K, R173A, Y175F, Y175W, Q17 8E, Q178K, T181T, T181A, T181D, T181G, T181N, T181R, T181S, T181E, P188E , P188G, P188N, P188S, P188D, P188K, P188R, P188T, G191C, Y205D, Y205N, N2 65G, E271K, E271L, N272R, N275E, N278D, N278E, N278G, N278K, F279Y, N280 V, H281C, H281F, H281I, H281L, H281T, H281V, H281M, V283L, Q291K, Q291R, A 294D, A294L, A294R, A294S, A294T, A294V, A294Y, A294E, A294K, A294N, A29 4Q, Q298A, Q298S, G301A, G301E, G301H, G301L, G301N, G301Q, G301R, G301S,G301T, G301V, G301Y, G301K, Y302A, Y302G, Y302L, Y302V, Y302W, Y302F, L307L, L307I, N309R, N309A, N309H, N309Q, N309D, N30 9E, V312L, S314A, K315E, K315T, K315V, H316A, H316E, H316F, H316L, H316M, H316Q, H316R, H316V, H316W, H316E, H316Y, L318Q, L 318D, L318E, K319L, S320A, T338P, T338Q, T338R, Q340D, Q340K, Q340M, Q340N, Q340R, Q340A, Q340E, Q340P, S356D, S356E, D372P, D372S, S373G, S373L, S373T, Q374D, Q374S, R375D, R375K, R375N, R375T, H406A, H406D, H406P, H406R, and H406K, wherein the amino acid positions correspond to the positions in SEQ ID NO:

1. 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 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with any one of the following: amino acid sequences shown in any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or their mature polypeptides. The mutant according to any one of claims 1-4 comprises substitutions at two, three, or four positions selected from amino acid positions 156, 205, 265, and 320; and the mutant does not contain any combination of substitutions selected from H205Y / Y156W and H205C / H156Y; Preferably, the substitution at amino acid position 156 is 156F, 156H or 156T; The substitution at amino acid position 205 is: 205D or 205N; The substitution at amino acid position 265 is: 265G; and / or The substitution at amino acid position 320 is: 320A. The mutant of claim 5 further comprises substitutions at one, two, three, four, or five positions of amino acid positions 123, 127, 128, 134, and 181; Preferably, the substitution at position 123 of the amino acid is: 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123F, 123L, 123N, 123T or 123Y; The substitutions at amino acid position 127 are: 127H, 127Q, 127E, or 127K; The substitutions at amino acid position 128 are: 128M, 128Q, 128R, 128T, 128A, 128D, 128E, 128I, 128K, 128L, or 128P; The substitutions at amino acid position 134 are: 134A, 134F, 134Q, 134S, 134V, 134D, 134E, 134K, 134M, 134N, 134P, 134T, or 134W; and / or The substitutions at amino acid position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E. The mutant as described in any one of claims 5-6 comprises a group of substitutions selected from any one of the following: 156H / 320A; 156H / 205D; 156H / 265G; 205D / 265G; 205D / 320A; 265G / 320A; 156H / 205D / 265G; 156H / 205D / 320A; 205D / 265G / 320A; 156H / 205D / 265G / 320A; 156H / 205D / 265G / 320A / 127K; 156H / 205D / 265G / 320A / 127K / 123N; 156H / 205D / 265G / 320A / 127K / 181E; 156H / 205D / 265G / 320A / 127K / 123N / 181E; and 156H / 205D / 265G / 320A / 127K / 123N / 181E / 128E / 134K. The mutant according to any one of claims 5-7 further comprises substitutions at one or more of the following amino acid positions: 116, 123, 124, 126, 127, 128, 133, 134, 136, 140, 142, 148, 152, 178 and / or 181. Preferably, the substitution at amino acid position 116 is: 116D, 116K, 116L, 116N, 116Q, 116E or 116M; The substitutions at amino acid position 123 are: 123E, 123H, 123I, 123K, 123Q, 123R, 123T, 123V, 123Y or 123N; The substitution at amino acid position 124 is: 124N; The substitution at amino acid position 126 is: 126L or 126C; The substitutions at amino acid position 127 are: 127H, 127Q, 127K, or 127E; The substitutions at amino acid position 128 are: 128I, 128K, 128M, 128Q, 128R, 128T, 128E, 128A, 128D or 128P; The substitution at amino acid position 133 is: 133Y; The substitutions at amino acid position 134 are: 134A, 134F, 134Q, 134S, 134T, 134V, 134W, 134E, 134N, 134P, 134K, 134D or 134M; The substitution at amino acid position 136 is: 136A; The substitution at amino acid position 140 is: 140R; The substitution at amino acid position 142 is: 142D; The substitution at amino acid position 148 is: 148D or 148N; The substitution at amino acid position 152 is: 152S; The substitution at amino acid position 178 is: 178K; and / or The substitutions at amino acid position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E. The mutant according to any one of claims 5-8 further comprises substitutions at one or more of the following amino acid positions: 132, 175, 188, 278, 281, 294, 301, 302, 309, 316, 318, 340, 356 and / or 406. Preferably, the substitution at amino acid position 132 is: 132D, 132K, 132T, 132V, 132W, or 132P; The substitution at amino acid position 175 is: 175F or 175W; The substitutions at amino acid position 188 are: 188E, 188G, 188N, 188S, 188K, or 188T; The substitutions at amino acid position 278 are: 278K, 278D, 278G, or 278E; The substitutions at amino acid position 281 are: 281C, 281F, 281I, 281L, 281T, 281V or 281M; The substitutions at amino acid position 294 are: 294D, 294E, 294L, 294R, 294S, 294T, 294V, 294Y, 294N, 294Q or 294K; The substitutions at amino acid position 301 are: 301S, 301T, 301V, 301Y, or 301K; The substitutions at amino acid position 302 are: 302A, 302G, 302L, 302V, 302W, or 302F; The substitutions at amino acid position 309 are: 309R, 309A, 309H, 309Q, 309D, or 309E; The substitutions at amino acid position 316 are: 316A, 316E, 316F, 316L, 316M, 316Q, 316R, 316V, 316W, 316E or 316Y; The substitutions at amino acid position 318 are: 318E, 318Q, or 318D; The substitutions at amino acid position 340 are: 340A, 340D, 340K, 340M, 340N, 340R, 340E, or 340P; The substitution at amino acid position 356 is: 356D or 356E; and / or The substitutions at amino acid position 406 are: 406A, 406D, 406P, 406R, or 406K. The mutant according to any one of claims 5-9, comprising the following substitutions relative to the amino acid sequence shown in SEQ ID NO:2: A123N / D124N / N126L / H133Y / K136A / H140R / H142D / S148D / D152S / Y156H / Q178K / T181E / Y205D / N265G / S320A. The mutant according to any one of claims 5-10, comprising, relative to the amino acid sequence shown in SEQ ID NO:2, a group of substitutions selected from any one of the following: T116E / A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134M / K136A / H140R / H142D / S148D / D152S / Y156H / Q178K / T181E / Y205D / N265G / S320A; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340P / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134D / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / E271K / N278D / H281M / A294K / G301K / Y302F / N309D / K315E / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278G / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / D124N / N126L / R127K / V128E / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294N / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128P / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294Q / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340P / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128A / E132P / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340P / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278E / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134K / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309E / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127E / V128E / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134D / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309E / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134N / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128A / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188K / Y205D / N265G / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128E / E132W / H133Y / R134P / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / K315T / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / D124N / N126L / R127K / V128D / E132W / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156H / Y175F / Q178K / T181E / P188T / Y205D / N265G / N278D / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123K / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / A294L / G301N / Y302W / L308F / N309A / H316A / L318E / S320A / Q340D / S356D / H406P; T116Q / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316Y / L318D / S320A / Q340A / S356D / H406K; T116M / A123N / N126C / R127K / V128E / E132P / R134K / Y156H / Y175W / T181E / G191C / Y205D / N265G / E271L / N278K / H281F / A294K / G301K / N309D / H316Y / L318D / S320A / Q340A / S356D / H406K; T116E / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281V / A294L / G301N / Y302W / L308F / N309E / H316Q / L318E / S320A / Q340E / S356D / H406P; T116E / A123N / N126C / R127H / V128E / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281M / A294Y / G301R / Y302V / L308F / N309E / H316A / L318E / S320A / Q340D / S356D / H406P; T116E / A123K / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281M / A294V / G301V / Y302W / L308F / N309E / H316Q / L318E / S320A / Q340D / S356D / H406P; T116E / A123Y / N126C / R127H / V128E / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281I / A294Q / G301K / Y302V / L308F / N309E / H316Y / L318E / S320A / S356D / H406P; T116E / A123Q / N126C / R127K / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281M / G301K / Y302W / L308F / H316R / L318E / S320A / Q340E / S356D / H406P; T116E / A123Y / N126C / R127K / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281V / A294Y / G301T / Y302A / L308F / N309E / L318E / S320A / Q340K / S356D / H406P; T116E / A123Y / N126C / V128K / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281I / A294K / G301E / Y302L / L308F / N309D / L318E / S320A / Q340K / S356D / H406P; T116E / A123Y / N126C / R127K / V128Q / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281M / A294L / G301R / Y302W / L308F / N309E / L318E / S320A / Q340A / S356D / H406P; T116E / A123K / N126C / V128I / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281V / G301N / Y302V / L308F / N309E / H316R / L318E / S320A / Q340D / S356D / H406P; T116E / A123E / N126C / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281I / A294T / G301K / Y302V / L308F / N309E / H316Q / L318E / S320A / Q340A / S356D / H406P; T116E / A123N / N126C / V128T / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281V / A294L / G301L / Y302L / L308F / N309E / H316Q / L318E / S320A / Q340D / S356D / H406P; T116E / A123R / N126C / R127K / V128I / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274V / N278K / H281I / A294E / G301K / Y302W / L308F / N309E / H316L / L318E / S320A / Q340A / S356D / H406P; T116E / A123N / N126C / R127Q / V128A / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281F / G301N / Y302W / L308F / N309E / H316Y / L318E / S320A / Q340K / S356D / H406P; T116E / A123H / N126C / V128E / E132P / R134K / Y156H / T181G / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281M / A294L / G301T / Y302G / L308F / N309E / H316A / L318E / S320A / Q340D / S356D / H406P; T116E / A123N / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / A294D / G301N / Y302W / L308F / N309Q / H316A / L318E / S320A / Q340K / S356D / H406A; T116E / A123E / N126C / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281I / A294L / Y302V / L308F / N309E / H316R / L318E / S320A / Q340A / S356D / H406P; T116E / A123R / N126C / V128M / E132P / R134K / Y156H / T181N / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294S / G301A / Y302W / L308F / N309E / L318E / S320A / Q340D / S356D / H406A; T116E / A123H / N126C / V128Q / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281C / A294V / G301T / Y302W / L308F / N309E / H316F / L318E / S320A / Q340D / S356D / H406P; T116E / A123K / N126C / R127K / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281I / A294L / G301N / Y302V / L308F / N309E / H316R / L318E / S320A / Q340K / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / R134S / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281F / A294K / G301Q / N309D / H316Y / L318D / S320A / Q340A / S356D / H406K; T116E / A123N / N126C / R127K / V128E / E132V / R134K / Y156H / T181E / G191C / Y205D / N265G / E271L / N278K / H281F / G301K / N309D / H316Y / L318D / S320A / Q340A / S356D / H406K; T116D / A123N / N126C / R127K / E132V / R134K / Y156H / Y175F / T181E / P188E / G191C / Y205D / N265G / E271L / N278K / H281F / A294K / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116N / A123N / N126C / R127K / V128D / E132V / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281F / G301T / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128D / E132P / R134E / Y156H / Y175F / P188T / G191C / Y205D / N265G / E271L / N278K / H281F / A294K / G301K / N309D / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281L / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123K / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340P / S356D / H406P; T116D / A123R / N126C / R127K / V128E / E132V / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116L / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116K / A123E / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116L / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116E / A123E / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406K; T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / H316V / L318E / S320A / Q340A / S356D / H406P; T116E / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / N309E / H316V / L318D / S320A / Q340E / S356D / H406K; T116E / A123K / N126C / R127K / V128E / E132P / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294L / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116M / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116K / A123K / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281L / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116K / A123N / N126C / R127K / V128E / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116E / A123K / N126C / R127K / V128E / E132P / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318D / S320A / Q340E / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294E / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116E / A123N / N126C / R127K / V128I / E132T / R134K / Y156H / T181N / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294N / G301Y / Y302F / L308F / N309D / H316Y / L318E / S320A / Q340K / S356D / H406A; T116E / A123N / N126C / V128I / E132K / R134K / Y156H / T181S / P188S / G191C / Y205D / N265G / E271L / N278K / H281M / A294N / G301A / Y302F / L308F / N309E / H316Y / L318E / S320A / S356E / H406P; T116E / A123I / N126C / V128M / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / A294Y / G301Q / L308F / N309E / H316M / L318E / S320A / Q340K / S356D / H406P; T116E / A123K / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340E / S356D / H406P; T116D / A123N / N126C / R127K / V128R / E132V / R134Q / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406K; T116D / A123N / N126C / R127K / V128E / E132V / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340A / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123R / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116E / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406K; T116E / A123K / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340A / S356D / H406P; T116L / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318D / S320A / S356D / H406P; T116D / A123N / N126C / R127K / V128E / E132V / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116D / A123K / N126C / R127K / V128E / E132V / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116L / A123K / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116E / A123N / N126C / R127K / V128E / E132P / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406K; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294L / G301K / Y302F / N309D / H316V / L318E / S320A / Q340E / S356D / H406P; T116E / A123K / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132P / R134W / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318D / S320A / Q340E / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116L / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / H316V / L318D / S320A / Q340A / S356D / H406P; T116K / A123E / N126C / R127K / V128E / E132V / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132P / R134W / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123N / N126C / R127K / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / L308M / N309E / H316V / L318E / S320A / Q340A / S356D / H406P; T116K / A123N / N126C / R127K / V128E / E132P / R134E / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / H316V / L318E / S320A / Q340E / S356D / H406K; T116E / A123K / N126C / V128E / E132P / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281M / A294E / G301R / Y302V / L308F / N309E / H316F / L318E / S320A / Q340M / S356D / H406P; T116E / A123N / N126C / V128E / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / G301Y / Y302W / L308F / N309E / H316M / L318E / S320A / Q340R / S356D / H406P; T116E / A123K / N126C / V128T / E132P / R134K / Y156H / T181A / P188S / G191C / Y205D / N265G / E271L / L274M / N278K / H281V / A294K / G301S / Y302F / L308F / N309E / H316M / L318E / S320A / Q340R / S356D / H406P; I49Y / T116M / A123K / N126C / R127K / V128M / E132P / R134P / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; A39C / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / N265G / E271L / N278K / H281M / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P; A39C / I49V / T116M / A123K / N126C / R127Q / V128E / E132P / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P; T116M / A123K / N126C / R127Q / V128E / E132P / R134T / S148N / Y156H / Y175F / T181E / G191C / Y205N / I212L / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K; A39C / I49Y / T116M / A123K / N126C / R127K / V128M / E132P / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; A39T / T116M / A123K / N126C / R127K / V128M / E132P / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; A39T / I49Y / T116M / A123K / N126C / R127Q / V128E / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294V / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K; I49Y / T116M / A123K / N126C / R127Q / V128M / E132P / Y156H / Y175F / T181E / G191C / Y205N / I212L / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P / S417P; A39C / I49Y / T116M / A123K / N126C / R127Q / V128M / E132P / R134T / Y156H / Y175F / T181E / G191C / Y205N / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K; A39T / T116M / A123K / N126C / R127H / V128E / E132P / R134T / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P; T116E / A123E / N126C / V128I / E132K / R134K / Y156H / T181D / P188N / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301T / Y302L / L308F / N309E / H316Q / L318E / S320A / Q340N / S356D / H406P; T116E / A123T / N126C / R127H / V128I / E132P / R134K / Y156H / Y175F / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281V / A294Q / G301N / Y302F / L308F / N309D / H316F / L318E / S320A / Q340D / S356D / H406P; T116E / A123K / N126C / R127K / V128M / E132K / R134T / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301S / Y302W / L308F / N309E / H316Q / L318E / S320A / Q340D / S356D / H406P; T116E / A123N / N126C / R127E / V128T / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / N278K / H281I / A294Q / G301S / Y302F / L308F / N309D / H316E / L318E / S320A / Q340K / S356D / H406D; T116E / A123T / N126C / V128E / E132T / R134K / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / N278K / H281I / A294R / G301N / Y302F / L308F / N309E / H316Y / L318Q / S320A / Q340A / S356D / H406A; T116E / A123N / N126C / R127K / V128E / E132V / R134K / Y156H / T181S / P188N / G191C / Y205D / N265G / E271L / N278K / H281L / A294Q / G301V / Y302V / L308F / N309E / H316Q / L318Q / S320A / Q340D / S356E / H406P; T116E / A123E / N126C / R127K / V128R / E132T / R134E / Y156H / T181S / P188E / G191C / Y205D / N265G / E271L / N278K / H281M / A294E / G301R / Y302A / L308F / N309E / V313L / H316Y / L318E / S320A / Q340R / S356D / H406A; T116E / A123V / N126C / R127H / V128Q / E132P / R134K / Y156H / T181S / P188G / G191C / Y205D / N265G / E271L / L274M / N278K / H281T / A294E / G301R / Y302G / L308F / N309E / H316Y / L318E / S320A / Q340E / S356D / H406P; T116E / A123N / N126C / V128T / E132P / R134V / Y156H / T181R / P188T / G191C / Y205D / N265G / E271L / N278K / H281I / A294E / G301R / Y302W / L308F / N309A / V313L / H316F / L318E / S320A / Q340E / S356D / H406P; T116E / A123E / N126C / V128E / E132P / R134K / Y156H / T181N / P188S / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301N / Y302W / L308F / N309H / V313L / H316W / L318D / S320A / Q340D / S356D / H406P; T116E / A123N / N126C / R127K / V128I / E132P / R134T / Y156H / T181N / P188N / G191C / Y205D / N265G / E271L / N278K / H281M / A294D / G301R / Y302A / L308F / L318E / S320A / Q340N / S356D / H406P; T116E / A123Y / N126C / R127E / V128I / E132D / R134A / Y156H / T181N / P188G / G191C / Y205D / N265G / E271L / L274I / N278K / H281V / A294V / G301S / Y302F / L308F / N309E / H316F / L318E / S320A / Q340E / S356D / H406P; T116E / A123E / N126C / V128I / E132T / R134T / Y156H / T181N / P188E / G191C / Y205D / N265G / E271L / L274I / N278K / H281M / A294D / G301N / Y302F / L308F / N309E / V313L / H316Q / L318E / S320A / Q340E / S356D / H406P; K23M / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; L61M / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; K106L / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / R134M / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / K154Y / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156F / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172E / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242L / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / K276S / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301H / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / K306H / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / D407E; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / R456L; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242F / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406R; W114M / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / K306Y / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; W114M / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301R / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; W114F / T116M / A123N / N126C / R127K / V128E / E132P / K154R / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172W / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302F / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; W114L / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / R456V; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301R / Y302F / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; W114L / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294N / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; W114F / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / D407R; T116M / A123N / N126C / R127K / V128E / E132P / K154L / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302W / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302W / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / R456L; Y62W / T116M / A123N / N126C / R127K / V128E / E132P / R134F / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; Y62W / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / K306Y / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302W / N309D / H316V / L318D / S320A / Q340A / S356D / H406P / D407R; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172K / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / K306R / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; Y62W / T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172L / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / R172K / Y175F / T181E / G191C / Y205D / R242F / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242T / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242S / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242A / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242S / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242T / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242N / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; T116M / A123N / N126C / R127K / V128E / E132P / Y156H / Y175F / T181E / G191C / Y205D / R242A / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370R / H406P; K23S / T116M / A123N / N126C / R127K / V128E / E132P / R134E / K154T / W155Y / Y156H / Y175F / T181E / G191C / Y205D / R242E / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370Q / H406P; K23Q / R74A / T116M / A123N / N126C / R127K / V128E / E132P / R134V / Y156H / Y175F / T181E / G191C / Y205D / R242E / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / K370H / H406P; I49A / T116M / A123K / N126C / R127Q / V128M / E132P / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K; T116M / A123N / N126C / R127H / V128E / E132P / R134T / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P; A39T / I49Y / T116M / A123K / N126C / R127Q / V128I / E132P / Y156H / Y175F / T181E / G191C / Y205N / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K; I49V / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P; A39T / I49Y / T116M / A123K / N126C / R127K / V128I / E132P / R134T / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P; A39T / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / Y205D / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K; I49Y / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K; I49Y / T116M / A123K / N126C / R127Q / V128E / E132P / R134T / S148N / Y156H / Y175F / T181E / G191C / Y205N / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K; I49A / T116M / A123K / N126C / R127Q / V128E / E132P / R134T / S148N / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K; T116M / A123K / N126C / R127Q / V128M / E132P / R134T / Y156H / Y175F / T181E / G191C / Y205N / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K; A39C / I49Y / T116M / A123K / N126C / R127Q / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; A39C / I49A / T116M / A123N / N126C / R127H / V128E / E132P / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P; A39T / T116M / A123K / N126C / R127K / V128E / E132P / R134P / Y156H / Y175F / T181E / G191C / Y205N / I212L / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K; A39T / T116M / A123K / N126C / R127Q / V128E / E132P / Y156H / Y175F / T181E / G191C / I212L / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P; I49V / T116M / A123K / N126C / R127Q / V128M / E132P / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P / S417P; I49Y / T116M / A123N / N126C / R127Q / V128E / E132P / R134T / Y156H / Y175F / T181E / G191C / I212L / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406K / S417P; A39C / I49V / T116M / A123K / N126C / R127Q / V128I / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / N265G / E271L / N278K / H281M / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K / S417P; A39T / I49Y / T116M / A123K / N126C / R127H / V128E / E132P / Y156H / Y175F / T181E / G191C / T213K / N 265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; I49Y / T116M / A123K / N126C / R127Q / V128E / E132P / S148N / Y156H / Y175F / T181E / G191C / T213K / N265G / E271L / N278K / H281M / A294V / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406P; A39C / I49V / T116M / A123K / N126C / R127K / V128M / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / T 213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P; A39T / I49Y / T116M / A123K / N126C / R127H / V128E / E132P / S148N / Y156H / Y175F / T181E / G191C / Y205N / T213K / N265G / E271L / N278K / H281M / A294Y / G301K / N309A / H316V / L318D / S320A / Q340A / S356D / H406P; and A39C / I49Y / T116M / A123K / N126C / R127Q / V128E / E132P / S148N / Y156H / Y175F / T181E / G191C / I212L / N 265G / E271L / N278K / H281M / A294Y / G301K / N309D / H316V / L318D / S320A / Q340A / S356D / H406K / S417P. The mutant according to any one of claims 5-7 further comprises substitutions at one or more of amino acid positions 126, 191, 271 and 278, preferably 126C, 191C, 271L and 278K, respectively. The mutant of claim 12 further comprises substitutions at one or more of the following amino acid positions: 116, 123, 127, 128, 132, 134, 175, 181, 188, 281, 294, 301, 302, 309, 315, 316, 318, 340, 356 and / or 406; Preferably, the substitution at amino acid position 116 is: 116N, 116Q, 116M, 116D, 116E, 116K, 116L or 116R; The substitutions at amino acid position 123 are: 123E, 123H, 123I, 123K, 123Q, 123R, 123V, 123Y, 123N, 123F, 123L, 123T; The substitutions at amino acid position 127 are: 127E, 127H, 127Q, or 127K; The substitutions at amino acid position 127 are: 128I, 128K, 128M, 128Q, 128R, 128T, 128E, 128D or 128A; The substitutions at amino acid position 132 are: 132D, 132K, 132T, 132V, 132W, or 132P; The substitutions at amino acid position 134 are: 134A, 134F, 134P, 134Q, 134S, 134V, 134M, 134K, 134E, 134W or 134T; The substitution at amino acid position 175 is: 175F or 175W; The substitutions at amino acid position 181 are: 181T, 181A, 181D, 181G, 181N, 181R, 181S, or 181E; The substitutions at amino acid position 188 are: 188E, 188G, 188N, 188S, 188K, 188T, 188D, or 188R; The substitutions at amino acid position 281 are: 281C, 281F, 281I, 281L, 281T, 281V or 281M; The substitutions at amino acid position 294 are: 294D, 294E, 294L, 294N, 294R, 294S, 294T, 294V, 294Y, 294K or 294Q; The substitutions at amino acid position 301 are: 301A, 301E, 301H, 301L, 301N, 301Q, 301R, 301S, 301T, 301V, 301Y or 301K; The substitutions at amino acid position 302 are: 302A, 302G, 302L, 302V, 302W, or 302F; The substitutions at amino acid position 309 are: 309R, 309A, 309H, 309Q, 309E, or 309D; The substitution at amino acid position 315 is: 315V; The substitutions at amino acid position 316 are: 316A, 316E, 316F, 316L, 316M, 316Q, 316R, 316V, 316W, 316E or 316Y; The substitutions at amino acid position 318 are: 318E, 318Q, or 318D; The substitutions at amino acid position 340 are: 340D, 340K, 340M, 340N, 340R, 340E, 340P, or 340A; The substitution at amino acid position 356 is: 356D or 356E; and / or The substitutions at amino acid position 406 are: 406A, 406D, 406P, 406R, or 406K. The mutant of claim 12 or 13, wherein the amino acid sequence represented by SEQ ID NO:2 comprises a group of substitutions selected from any one of the following: N265G / E271L / N126C / S320A / Y156H / Y205D / N278K / G191C; and N265G / E271L / N126C / S320A / Y156H / Y205D / N278K / G191C / R127K / H406K / H281M / L318D / G301K / T181E / H316Y / Y175F / E132W / S356D / K315V / A294K. The mutant according to any one of claims 12-14, comprising, relative to the amino acid sequence shown in SEQ ID NO:2, a group of substitutions selected from any one of the following: N126C / G191C / S320A / Y156H / H205D / N265G; T116D / A123L / N126C / R127K / V128E / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116K / A123N / N126C / R127K / E132W / R134W / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / N126C / R127K / V128D / E132W / R134M / Y156H / Y175F / T181E / G191C / Y205D / N265G / E27 1L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / N126C / R127K / V128E / E132P / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340P / S356D / H406K; T116E / A123N / N126C / R127K / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340P / S356D / H406K; T116K / A123F / N126C / R127K / V128E / E132W / R134W / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116L / A123N / N126C / R127K / V128E / E132W / R134W / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / H316Y / L318D / S320A / Q340E / S356D / H406K; T116E / A123N / N126C / R127K / V128E / E132W / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309D / K315V / H316Y / L318D / S320A / Q340P / S356D / H406K; T116D / A123N / N126C / R127K / E132W / R134K / Y156H / Y175F / T181E / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / N126C / R127K / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309D / K315V / H316Y / L318D / S320A / Q340P / S356D / H406K; T116E / A123N / N126C / R127K / V128E / E132W / R134T / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309D / K315V / H316Y / L318D / S320A / Q340A / S356D / H406K; T116E / A123N / N126C / R127K / V128D / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / H316Y / L318D / S320A / Q340E / S356D / H406K; T116K / A123N / N126C / R127K / V128E / E132W / R134W / Y156H / Y175F / T181E / P188D / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116R / A123N / N126C / R127K / V128D / E132W / R134E / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116K / A123L / N126C / R127K / V128E / E132W / R134W / Y156H / Y175F / T181E / P188R / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / S356D / H406K; T116M / A123T / N126C / R127K / V128D / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356E / H406K; T116D / A123F / N126C / R127K / V128E / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / N126C / R127K / V128E / E132W / R134M / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294Q / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116D / A123N / N126C / R127K / V128D / E132W / R134K / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / N126C / R127K / V128A / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123F / N126C / R127K / V128D / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / N126C / R127K / V128A / E132W / R134E / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123F / N126C / R127K / V128A / E132W / R134E / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / N126C / R127K / V128A / E132W / R134M / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356E / H406K; T116D / A123N / N126C / R127K / V128A / E132W / R134K / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / N126C / R127K / V128E / E132W / R134M / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309D / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123F / N126C / R127K / V128A / E132W / R134E / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; T116M / A123N / N126C / R127K / V128D / E132W / R134E / Y156H / Y175F / T181E / P188T / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K; and T116M / A123N / N126C / R127K / V128E / E132W / R134E / Y156H / Y175F / T181E / P188K / G191C / Y205D / N265G / E271L / N278K / H281M / A294K / G301K / Y302F / N309E / K315V / H316Y / L318D / S320A / Q340E / S356D / H406K. The mutant according to any one of claims 5-7, wherein the amino acid sequence represented by SEQ ID NO:2 comprises a group of substitutions selected from any one of the following: N265G; Y205D; Y156H; S320A; Y156H / Y205D; Y155H / N265G; Y156H / S320A; Y205D / N265G; Y205D / S320A; N265G / S320A; 156H / 205D / 265G; 156H / 205D / 320A; 205D / 265G / 320A; S320A / Y156H / H205D / N265G; A48S / I49A / A52S / W68N / Y156H / Y205D / N265G / S320A; A48S / I49A / A52D / W68N / Y156H / Y205D / N265G / S320A; T116R / A123N / D124N / R127K / V128E / G131D / E132W / H133Y / R134M / Y156H / Y205D / N265G / S320A; T116E / A123N / D124N / R127E / V128I / G131E / E132P / H133Y / R134M / Y156H / Y205D / N265G / S320A; T116E / A123N / R127K / V128E / G131K / E132P / H133Y / R134T / Y156H / Y205D / N265G / S320A; T116D / A123N / R127K / G131D / E132W / H133F / R134K / Y156H / Y205D / N265G / S320A; T116E / A123N / R127K / V128I / G131D / E132A / H133F / R134M / Y156H / Y205D / N265G / S320A; T116M / A123N / R127K / V128E / G131E / E132P / H133Y / R134M / Y156H / Y205D / N265G / S320A; T116E / A123N / R127K / G131E / E132P / H133F / R134K / Y156H / Y205D / N265G / S320A; T116K / A123N / R127K / V128I / G131D / E132W / H133Y / R134N / Y156H / Y205D / N265G / S320A; T116W / A123N / R127K / V128E / E132W / H133Y / R134K / Y156H / Y205D / N265G / S320A; T116M / A123N / D124N / R127K / V128I / G131D / H133Y / R134M / Y156H / Y205D / N265G / S320A; T116E / A123N / R127K / V128E / G131E / E132W / H133Y / R134T / Y156H / Y205D / N265G / S320A; T116E / A123N / G131D / E132W / H133Y / R134T / Y156H / Y205D / N265G / S320A; T116E / A123N / D124N / R127K / V128I / G131D / E132W / H133Y / R134T / Y156H / Y205D / N265G / S320A; T116E / A123N / D124N / R127K / G131D / E132W / H133Y / R134K / Y156H / Y205D / N265G / S320A; T116R / A123N / D124N / R127K / V128E / G131D / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A; T116R / A123N / R127K / G131D / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A; T116K / A123N / D124N / R127K / V128L / G131D / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A; T116K / A123N / R127K / V128I / G131D / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A; T116K / A123Y / R127K / V128E / G131E / E132W / H133Y / R134E / Y156H / Y205D / N265G / S320A; A123N / R127K / V128L / G131E / E132P / H133F / R134K / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116K / A123N / D124N / R127K / G131E / H133Y / R134W / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116K / A123N / R127K / V128E / G131E / E132P / H133F / R134W / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116K / A123N / R127K / V128I / G131D / E132W / H133Y / R134D / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116E / A123N / D124N / R127K / V128I / G131E / E132P / H133Y / R134W / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A; T116R / A123N / R127K / G131D / E132W / H133Y / R134E / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116E / A123N / R127K / V128E / G131E / E132W / H133Y / R134K / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A; T116E / A123N / R127K / V128I / G131K / E132P / H133F / R134K / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A; A123N / R127K / V128I / G131E / H133Y / R134E / Y156H / Q178K / T181E / P188T / Y205D / N265G / S320A; T116E / A123N / D124N / R127E / V128I / G131D / E132W / H133Y / R134N / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A; T116D / A123N / R127K / G131E / E132K / H133Y / R134K / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; A123N / R127K / G131D / H133F / R134E / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A; T116M / A123N / R127K / V128I / G131E / E132P / H133Y / R134M / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; A123N / D124N / R127K / V128I / G131E / E132P / H133Y / R134K / Y156H / Q178E / T181E / P188T / Y205D / N265G / S320A; T116M / A123N / R127K / V128L / G131E / E132P / H133Y / R134K / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116K / A123F / R127K / V128D / G131E / E132P / H133Y / R134E / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116E / A123N / R127K / V128D / G131E / E132W / H133Y / R134K / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A; T116K / A123Y / R127K / V128D / G131E / H133Y / R134E / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A; T116E / A123N / D124N / R127K / V128L / G131K / E132P / H133Y / R134K / Y156H / Q178K / T181E / P188K / Y205D / N265G / S320A; A123F / D124N / R127K / V128D / G131K / E132P / H133Y / R134K / Y156H / Q178E / T181E / P188T / Y205D / N265G / S320A; T116K / A123N / R127K / V128L / G131D / E132A / H133Y / R134E / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116M / A123N / R127K / V128K / G131E / E132P / H133Y / R134M / Y156H / Q178E / T181E / P188K / Y205D / N265G / S320A; T116E / A123Y / R127K / V128D / G131E / E132P / H133F / R134T / Y156H / Q178K / T181E / P188T / Y205D / N265G / S320A; T116M / A123N / D124N / R127K / V128L / G131E / H133Y / R134E / Y156H / Q178K / T181E / P188T / Y205D / N265G / S320A; Y156H / Y205D / N265G / E271K / N272R / N275E / N278E / F279Y / N280V / H281M / V283L / Q291K / A294E / Q298A / G301K / L307I / N309D / V312L / S314A / K315E / L318E / K319L / S320A; Y156H / Y205D / N265G / N272R / N275E / N278D / F279Y / N280V / H281M / Q291R / A294E / Q298S / G301K / L307I / N309D / V312L / S314A / L318D / K319L / S320A; Y156H / Y205D / N265G / S320A / D372P / S373T / Q374D / R375K; Y156H / Y205D / N265G / S320A / T338Q / D372P / S373G / Q374D / R375K; Y156H / Y205D / N265G / S320A / D372P / S373L / Q374S / R375N; Y156H / Y205D / N265G / S320A / D372S / S373G / Q374S / R375D; Y156H / Y205D / N265G / S320A / D372P / S373G / Q374S / R375K; Y156H / Y205D / N265G / S320A / D372P / S373L / Q374D / R375T; Y156H / Y205D / N265G / S320A / T338R / D372P / S373T / Q374S / R375D; Y156H / Y205D / N265G / S320A / T338P / D372P / S373T / Q374D / R375N; and Y156H / Y205D / N265G / S320A / D372P / S373T / Q374S / R375N. The mutant according to any one of claims 1-4 comprises substitutions at one or more of the amino acid positions 123, 127, 128, 134, 136, 142, 148, 178 and / or 181; Preferably, the substitution at amino acid position 123 is 123N; The substitution at amino acid position 127 is: 127K; The substitution at amino acid position 128 is either 128E or 128I. The substitution at amino acid position 134 is: 134E, 134K or 134M; The substitutions at amino acid position 136 are: 136A, 136D, or 136E; The substitution at amino acid position 142 is: 142D; The substitution at amino acid position 148 is either 148T or 148D. The substitution at amino acid position 178 is: 178K or 178E; and / or The substitution at amino acid position 181 is: 181E. The mutant of claim 17 further comprises substitutions at one or more of the amino acid positions 126, 191, 271, and 278, preferably 126C, 191C, 271L, and 278K, respectively. The mutant of claim 17 or 18, wherein the amino acid sequence represented by SEQ ID NO:2 comprises a group of substitutions selected from any one of the following: A123N / T181E / H142D / R127K; A123N / T181E / H142D / R127K / K136N; and A123N / T181E / H142D / R127K / K136N / Q178K. The mutant according to any one of claims 17-19, comprising, relative to the amino acid sequence shown in SEQ ID NO:2, a group of substitutions selected from any one of the following: A123N / N126L / R127K / V128E / E132P / H133Y / R134K / K136A / H140K / H142D / S148T / Y156H / Q178E / T181E / Y205D / N265G / S320A; A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134K / K136A / H140R / H142D / S148T / Y156H / Q178E / T181E / Y205D / N265G / S320A; A123N / D124N / N126L / R127K / V128E / H133Y / R134E / K136A / H140R / H142D / S148D / D152S / Y156T / Q178K / T181E; T116E / A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134M / K136A / H140R / H142D / S148D / D152S / Y156T / Q178K / T181E; A123N / N126L / R127K / V128E / E132P / H133Y / R134K / K136A / H140K / H142D / S148T / Y156T / Q178E / T181E; A123N / N126L / R127K / V128E / H133Y / R134E / K136A / H140R / H142D / S148D / Y156T / Q178K / T181E; A123N / D124N / N126L / R127K / V128E / E132P / H133Y / R134K / K136A / H140R / H142D / S148T / Y156T / Q178E / T181E; T116D / A123N / N126L / R127K / V128E / E132W / H133Y / R134K / K136A / H140K / H142D / S148D / D152S / Y156T / Q178K / T181E; V101L / A111S / T116M / A123N / R127K / V128I / G131E / R134E / K136A / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178K / T181E; V101L / A111S / T116M / V118Q / A123N / R127K / V128I / G131E / R134E / K136A / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178K / T181E; V101L / A111S / T116K / A123N / R127K / V128I / G131E / R134E / K136A / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178E / T181E; V101L / A111S / W114T / T116M / A123N / R127K / V128I / G131D / R134E / K136E / H14 2D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178K / T181E; V101L / A111S / W114E / T116M / A123N / R127K / V128I / G131D / R134M / K136D / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R172K / R173A / Q178K / T181E; and V101L / A111S / T116M / V118Q / A123N / R127K / V128I / G131E / R134E / K136A / H142D / S148T / E167Y / S168K / R169T / K170N / L171K / R173A / Q178K / T181E. The polynucleotide encoding the α-amylase mutant according to any one of claims 1-20. A recombinant expression vector comprising the polynucleotide of claim 21. A host cell comprising the polynucleotide of claim 21 or the recombinant expression vector of claim 22. The host cell as described in claim 23 is a bacterium. The host cell of claim 24, wherein the bacteria are derived from the genus Bacillus. The host cell as described in any one of claims 23-25 ​​is Bacillus subtilis or Bacillus licheniformis. The method for preparing the α-amylase mutant according to any one of claims 1-20 comprises the following steps: (a) Culture the host cells according to any one of claims 23-26 under conditions suitable for the expression of the α-amylase mutant; (b) Recover the expressed α-amylase mutant. The α-amylase mutant obtained by the method of claim 27. A composition comprising the α-amylase mutant according to any one of claims 1-20 and one or more other enzymes. The composition of claim 29, 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. Use of the α-amylase mutant according to any one of claims 1-20 or the composition according to any one of claims 29-30 for liquefying starch-containing materials. Use of the α-amylase mutant according to any one of claims 1-20 or the composition according to any one of claims 29-30 for washing. Use of the α-amylase mutant according to any one of claims 1-20 or the composition according to any one of claims 29-30 for desizing textiles. Use of the α-amylase mutant according to any one of claims 1-20 or the composition according to any one of claims 29-30 for producing baked goods.

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