Glucoamylase variants and polynucleotides encoding them

Abstract

The present invention relates to glucoamylase variants having increased specific activity and / or an increased melting temperature, measured by TSA, of at least 0.5 degrees Celsius, and further wherein said variants have at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the amino acid sequence of SEQ ID NO: 3. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680058352.X, filed on October 12, 2016, entitled "Glucosylamylase variants and polynucleotides encoding them".

[0002] References to sequence lists This application contains a sequence list in computer-readable form, which is incorporated herein by reference. Background of the Invention Technical Field

[0003] This invention relates to glucose amylase variants, polynucleotides encoding these variants, methods for producing these variants, and methods for using these variants. The use of the glucose amylase of the present invention for starch conversion to produce fermentation products (e.g., ethanol) and syrups (e.g., glucose) is also described. The invention further relates to compositions comprising the glucose amylase variants of the present invention.

[0004] Related fields description Glucoamylase (1,4-α-D-glucan glucosylhydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts, with those from the genus *Aspergillus* being the most commercially important.

[0005] Commercially, glucosylamylase is used to convert starch-containing materials that have been partially hydrolyzed by α-amylase into glucose. The glucose can then be converted directly or indirectly into fermentation products using a fermenting organism. Examples of commercial fermentation products include alcohols (e.g., ethanol, methanol, butanol, 1,3-propanediol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid, gluconate, lactic acid, succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); and more complex compounds, including, for example, antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B vitamins). 12 Fermentation processes are also commonly used in the edible alcohol (e.g., beer and wine) industry and the dairy (e.g., in the production of yogurt and cheese) industry.

[0006] The final product can also be a syrup. For example, the final product can be glucose, but it can also be converted into fructose or a mixture of nearly equal amounts of glucose and fructose by glucose isomerase. Such a mixture, or a mixture further enriched with fructose, is the most commonly used commercially available high-fructose corn syrup (HFCS) worldwide.

[0007] The object of this invention is to provide polypeptides with glucosylamylase activity and polynucleotides encoding these polypeptides, and these polypeptides and polynucleotides provide high yields in the production of fermentation products (e.g., ethanol production processes).

[0008] WO 2011 / 068803 discloses glucosyl amylase isolated from the fungus Myxophytes, particularly Myxophytes squarrosa and Myxophytes squarrosa.

[0009] This invention provides a glucose amylase variant with improved properties compared to its parent.

[0010] WO 2014 / 177546 and WO 2016 / 062875 disclose *Cladosporium* species with increased heat stability and increased specific activity. Gloeophyllum trabeum ) a variant of glucosylamylase. Summary of the Invention

[0011] In a first aspect, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, I13, K15, V18, L19, N25, S27, K28, S30, V36, V37, T43, D45, S57, V59, F60, I71, S73, T74, L77, D82, D83, V85, T86, E88, L91, S95, P97, T103, D114, S134, L137, T139, N142, L145, S146, N147, N149, Y152, V153, T154, S1 55. N156, L157, W158, P159, I160, Q162, V169, S170, S175, T176, Y177, D184 , S186, R199, A202, A203, T206, Q210, T211, S212, Q213, V214, S215, Y217, T 218, T219, Q220, A221, D222, N223, L224, F227, Y231, P234, S235, Y238, T24 0. T243, G244, G245, G246, R247, S248, A252, T254, L255, Y262, S265, G267, A270, A271, K279, S282, L284, V294, Y295, S296, I297, N298, S299, G300, A 302, S303, N304, T309, E314, S316, Q318, G319, T326, V330, N339, E342, S34 3. E348, S351, T352, Q359, S362, G363, V364, T365, A366, S371, S372, T378, S381, I383, N385, F386, A392, N394, K396, Y408, K410, D412, S414, S417, V4 19. A426, S427, E433, A434, N436, N437, T438, Q439, G442, A446, L448, V450 , N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N 495, S501, A502, T506, I509, T510, N512, S516, A518, I519, N527, N528, A530, E534, D536, P537, N538, N539, I541, A545, S546, G547, S548, N552, and T554,The positions described therein correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more of these positions provide a glucosylamylase variant having an increase in melting temperature of at least 0.5 degrees Celsius as measured by TSA; and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0012] In a second aspect, the present invention relates to glucosylamylase variants comprising substitutions at one or more positions selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, G11, I13, K15, A16, V18, L19, N25, S27, S30, A32, A34, V36, V37, S44, S57, V59, F60, Y67, T68, I71, D72, S73, T74, S75, S76, L77, R78, D82, D83, F84, V85, T86, N90, L91, Q93, S95, L101, T102, T10 3. S134, L137, T139, N142, L145, S146, N147, Y152, V153, T154, S155, L157, W158, P159, I160, Q162, N163, S170, S175, T176, Y177, S186, R1 99. A202, A203, T206, Q210, T211, S212, Q213, S215, Q220, A221, D222, N223, L224, F227, P234, S235, Y238, T240, T243, G244, G245, G246, S 248, A252, T254, L255, A270, A271, K279, S282, L284, Y295, S296, I297, N298, S299, G300, A302, S303, N304, S316, G319, T326, V330, N339, E342, S343, Q344, E348, S351, Q359, S362, G363, T365, A366, S371, S372, T378, S381, I383, F386, A392, N394, K396, N401, K410, D412, S414 S417, V419, D420, E433, N437, T438, Q439, F440, G442, A446, N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N495, S501, A502, I509, T510, N512, S516, A518, I519, N527, A530, E534, P537, N538, N539, I541, A545, S546, G547, N552, and T554, wherein the positions correspond to in SEQ IDNO: 3 indicates the amino acid positions in the amino acid sequence; and the substitutions at one or more of these positions provide a glucosylamylase variant having a value of at least 1 as measured by the improvement factor IF.The specific activity of 1 is increased, and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0013] The present invention also relates to polynucleotides encoding variants of the invention; nucleic acid constructs, vectors, and host cells comprising said polynucleotides; and methods for generating said variants.

[0014] The present invention further relates to compositions comprising the variant glucosylamylase of the present invention.

[0015] In another aspect, the present invention relates to the use of this variant of glucosylamylase in the production of syrups or fermentation products.

[0016] In another aspect, the present invention relates to a method for producing fermentation products from starch-containing materials, comprising the following steps: (a) Liquefying starch-containing materials in the presence of an α-amylase; (b) Saccharifying the liquefied material; and (c) Fermentation is carried out using a fermenting organism; Step (b) is performed using at least one variant of the glucosyl amylase of the present invention.

[0017] In another aspect, the present invention relates to a method for producing fermentation products from starch-containing materials, comprising the following steps: (a) Saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of the starch-containing material; and (b) Fermentation using fermentation organisms. Step (a) is performed using at least one variant of the glucosyl amylase of the present invention.

[0018] In a further embodiment, the present invention relates to a method for producing a syrup product from a starch-containing material, the method comprising the steps of: (a) liquefying the starch-containing material in the presence of an α-amylase; and (b) saccharifying the liquefied material in the presence of a variant of glucosylamylase of the present invention.

[0019] In another embodiment, the present invention relates to a method for producing a syrup product from a starch-containing material, the method comprising the steps of: saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of the starch-containing material in the presence of a variant of the glucosyl amylase of the present invention.

[0020] definition Glucoamylase: The term "glucosylase" (1,4-α-D-glucan glucosylhydrolase, EC 3.2.1.3) is defined as an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules. For the purposes of this invention, glucosylase activity was determined according to the procedures described in the examples herein. Glucoamylase units (AGU) are defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute under standard conditions (37°C, pH 4.3, substrate: 23.2 mM maltose, buffer: 0.1 M acetate, reaction time: 5 min).

[0021] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (without alteration in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0022] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks intron sequences that can be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA, which undergoes a series of steps (including splicing) before becoming mature, spliced ​​mRNA.

[0023] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly identifies the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame (ORF), which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0024] Control Sequence: The term "control sequence" refers to the nucleic acid sequence necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding that variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to, pre-progenitors, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, as well as transcription and translation termination signals. Control sequences may be provided with adapters for the purpose of introducing specific restriction sites that facilitate the connection of control sequences to the coding regions of polynucleotides encoding variants.

[0025] Expression: The term “expression” includes any step involved in variant generation, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0026] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide that encodes a variant and is effectively linked to a control sequence provided for its expression.

[0027] Fragment: The term "fragment" refers to a polypeptide that has one or more (e.g., several) amino acids missing from the amino and / or carboxyl termini of a mature polypeptide; wherein the fragment has glucosylamylase activity.

[0028] Highly stringent conditions: The term "highly stringent conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0029] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using a nucleic acid construct or expression vector containing the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0030] Improved properties: The term "improved properties" refers to variant-related characteristics that are improved compared to the parent. Such improved properties include, but are not limited to, increased specific activity and increased thermal stability.

[0031] Isolated: The term “isolated” means a substance in a form or environment not naturally present. Non-limiting examples of isolated substances include (1) any substance not naturally present, (2) any substance, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more naturally present components naturally associated with it; (3) any substance artificially modified relative to a naturally found substance; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding the substance; use of a promoter stronger than that naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples.

[0032] Low stringency conditions: The term "low stringency conditions" refers to the following standard DNA blotting procedure for probes of at least 100 nucleotides in length: pre-hybridization and hybridization at 42 °C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 50 °C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0033] Mature polypeptide: 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. In one aspect, a mature polypeptide is amino acids 18 to 573 of SEQ ID NO: 2. Amino acids 1 to 17 of SEQ ID NO: 2 are signal peptides. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. Mature polypeptides are also included herein as SEQ ID NO: 3.

[0034] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide with glucosylamylase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 52 to 1719 of SEQ ID NO: 1. Nucleotides 1 to 51 of SEQ ID NO: 1 encode a signal peptide.

[0035] Medium-tough conditions: The term "medium-tough conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0036] Medium-high stringent conditions: The term "medium-high stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0037] Mutant: The term “mutant” refers to a polynucleotide that encodes a variant.

[0038] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a way that does not normally exist in nature to contain a segment of nucleic acid, or is synthesized and contains one or more control sequences.

[0039] Operablely linked: The term “operably linked” refers to a configuration in which a control sequence is positioned relative to the coding sequence of a polynucleotide so that the control sequence guides the expression of the coding sequence.

[0040] Parental or parental glucosylamylase: The term “parental” or “parental glucosylamylase” refers to any polypeptide having glucosylamylase activity, which is modified to produce the enzyme variant of the present invention.

[0041] Sequence consistency: The parameter “sequence consistency” is used to describe the correlation between two amino acid sequences or two nucleotide sequences.

[0042] For the purposes of this invention, the following methods are used: (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000) Trends Genet. [Trends in Genetics] The Needleman-Wunsch algorithm implemented in the Needle program (preferably version 5.0.0 or later) 16: 276-277 (Needleman and Wunsch, 1970) J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) was used to determine sequence identity between two amino acid sequences. The parameters used were a 10-point nick opening penalty, a 0.5-point nick expansion penalty, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output marked "Longest Consistency" (obtained using the –nobrief option) was used as the percentage consistency and calculated as follows: (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment) For the purposes of this invention, the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) implemented in the Niedle program, such as in the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 5.0.0 or later), is used to determine sequence consistency between two deoxyribonucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled “Longest Consistency” (obtained using the –nobrief option) is used as the percentage consistency and calculated as follows: (Consistent deoxyribonucleotides x 100) / (Alignment length - Total number of vacancies in the alignment) Variants: The term "variant" means a polypeptide having glucosylamylase activity that includes alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying the position. These variants of the present invention have at least 20%, for example, 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% glucosylamylase activity of the polypeptide of SEQ ID NO: 3.

[0043] Very High Tough Conditions: The term "very high tough conditions" means that for probes at least 100 nucleotides in length, standard DNA blotting procedures are followed, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0044] Very low stringency conditions: The term "very low stringency conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 45°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0045] Wild-type glucosylamylase: The term "wild-type" glucosylamylase refers to a glucosylamylase expressed by naturally occurring microorganisms, such as bacteria, yeasts, or filamentous fungi found in nature. In one embodiment, wild-type glucosylamylase is derived from *Entoloma viminalis* (…). Gloeophyllum sepiarium In this disclosure, it is also referred to as Gs-AMG.

[0046] Variant Naming Rules For the purposes of this invention, the polypeptide disclosed in SEQ ID NO: 3 was used to determine the corresponding amino acid residues in another glucosylamylase. The amino acid sequence of the other glucosylamylase was compared with the mature polypeptide disclosed in SEQ ID NO: 3, and based on this comparison, the amino acid sequence was determined using a software package such as EMBOSS (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000). Trends Genet. [Trends in Genetics] 16: 276-277 (preferably version 5.0.0 or later) implements the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, ) in the Niedle program. J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) was used to determine the amino acid position numbers corresponding to any amino acid residues in the polypeptide disclosed in SEQ ID NO: 3. The parameters used were a nick opening penalty of 10, a nick expansion penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0047] The identification of corresponding amino acid residues in another glucosylamylase can be determined by using several computer programs that compare multiple polypeptide sequences with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparisons by logarithmic prediction; version 3.5 or later; Edgar, 2004). Nucleic Acids Research [Nucleic Acid Research] 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research [Nucleic Acid Research] 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research [Nucleic Acid Research] 33: 511-518; Katoh and Toh, 2007. Bioinformatics [Bioinformatics] 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, BioinformaticsBioinformatics 26: 1899–1900) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research [Nucleic Acid Research] 22: 4673-4680.

[0048] When other enzymes deviate from the polypeptide of SEQ ID NO: 3, making it impossible for traditional sequence-based comparison methods to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. [Journal of Molecular Biology] 295:613-615), other pairwise sequence comparison algorithms can be used. Higher sensitivity in sequence-based searches can be achieved using search procedures that employ probabilistic representations (profiles) of peptide families to search a database. For example, the PSI-BLAST procedure generates multiple profiles through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997). Nucleic Acids Res. [Nucleic Acid Research] 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. Procedures such as GenTHREADER (Jones, 1999) can be used. J. Mol. Biol. [Journal of Molecular Biology] 287: 797-815; McGuffin and Jones, 2003, Bioinformatics [Bioinformatics] 19: 874-881) utilizes information from multiple sources (PSI-BLAST, secondary structure prediction, structure alignment spectra, and solvation potential) as input to a neural network that predicts the structural folding of a query sequence. Similarly, Gough et al., 2000, J. Mol. Biol The method described in [Journal of Molecular Biology] 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.

[0049] For proteins with known structures, several tools and resources are available for retrieving and generating structural alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms, such as distance alignment matrices (Holm and Sander, 1998), can be used. Proteins [Protein] 33: 88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering[Protein Engineering] 11: 739-747) compares two or more protein structures, and implementations of these algorithms can also be used to query structure databases containing structures of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics [Bioinformatics] 16: 566-567.

[0050] In the description of variations of the invention, the following nomenclature is provided for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are used.

[0051] Substitution For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg + Ser411Phe" or "G205R + S411F" indicates that glycine (G) and serine (S) at positions 205 and 411 are substituted with arginine (R) and phenylalanine (F), respectively.

[0052] Deletion For amino acid deletions, use the following nomenclature: initial amino acid, position, Therefore, the deletion of glycine at position 195 will be represented as "Gly195". "or "G195 Multiple missing items are separated by plus signs ("+"), for example, "Gly195". + Ser411 "or "G195 + S411 ".

[0053] Insertion For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, inserting lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, inserting lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0054] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be:

[0055] Multiple alterations Variants containing multiple changes are separated by a plus sign ("+"), such as "Arg170Tyr +Gly195Glu" or "R170Y + G195E", which indicate that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively.

[0056] Different alterations Where different changes can be introduced at a single position, these changes are separated by commas, for example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala + Arg170Gly,Ala" represents the following variant: “Tyr167Gly + Arg170Gly”, “Tyr167Gly + Arg170Ala”, “Tyr167Ala + Arg170Gly” and “Tyr167Ala + Arg170Ala”. Detailed Implementation

[0057] This invention relates to glucosylamylase variants having improved properties compared to their parental glucosylamylase. In specific embodiments, the parental glucosylamylase is a glucosylamylase derived from *Entoloma viminalis*, such as the glucosylamylase disclosed herein as SEQ ID NO: 3. In specific embodiments, the improved properties are selected from increased thermal stability, as measured by an increase in melting temperature as determined by TSA assay in Example 1. In another specific embodiment, the improved properties are selected from increased specific activity determined as an increased improvement factor (IF), as measured as the relative specific activity determined by acarbose assay as described in Example 1. The increase in IF is calculated relative to the wild-type enzyme of SEQ ID NO: 3 (which has IF = 1.0).

[0058] variants This invention provides glucosylamylase variants comprising substitutions at one or more positions selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, I13, K15, V18, L19, N25, S27, K28, S30, V36, V37, T43, D45, S57, V59, F60, I71, S73, T74, L77, D82, D83, V85, T86, E88, L91, S95, P97, T103, D114, S134, L137, T139, N142, L145, S146, N147, N149, Y152, V153, T154, S155, N1 56. L157, W158, P159, I160, Q162, V169, S170, S175, T176, Y177, D184, S18 6. R199, A202, A203, T206, Q210, T211, S212, Q213, V214, S215, Y217, T218, T219, Q220, A221, D222, N223, L224, F227, Y231, P234, S235, Y238, T240, T 243, G244, G245, G246, R247, S248, A252, T254, L255, Y262, S265, G267, A27 0. A271, K279, S282, L284, V294, Y295, S296, I297, N298, S299, G300, A302 , S303, N304, T309, E314, S316, Q318, G319, T326, V330, N339, E342, S343, E 348, S351, T352, Q359, S362, G363, V364, T365, A366, S371, S372, T378, S3 81. I383, N385, F386, A392, N394, K396, Y408, K410, D412, S414, S417, V419 , A426, S427, E433, A434, N436, N437, T438, Q439, G442, A446, L448, V450, N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N4 95, S501, A502, T506, I509, T510, N512, S516, A518, I519, N527, N528, A530, E534, D536, P537, N538, N539, I541, A545, S546, G547, S548, N552, and T554,The positions described therein correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more of these positions provide a glucosylamylase variant having an increase in melting temperature of at least 0.5 degrees Celsius as measured by TSA; and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0059] In a specific embodiment, the glucosylamylase variant comprises substitutions at one or more positions selected from the group consisting of: Q1K, Q1R, S2E, S2K, S2L, S2P, S2R, V3L, V3G, V3R, D4R, D4S, D4G, D4A, D4W, S5L, S5V, S5G, S5C, S5R, S8Q, S8H, S8A, S8Y, S9C, S9Q, S9M, S9W, S9D, S9G, I13V, I13R, I13S, I13L, I13E, K15G, K15R, V18M, V18Q, L19G, L19F, N25S, N25A, S27A, S27L, S27G, S27 V, S27C, K28C, K28R, S30Q, S30A, S30K, S30T, S30L, V36K, V36G, V36W, V36A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, D45L, D45P, S57 P, S57L, S57G, S57F, S57R, S57T, S57A, V59G, V59T, V59S, V59E, F60S, I71M, I71S, I71T, I71V, S73H, S73A, S73R, S73N, S73V, S73G, T74V, L77S, L77P, L77 R, D82N, D82R, D82V, D82G, D83L, D83C, D83W, V85Q, V85G, V85P, T86R, T86V, E88Q, E88R, E88G, L91S, L91P, L91G, S95A, S95P, S95T, S95V, P97T, P97I, P9 7R, T103Y, T103A, T103G, D114G, D114N, D114M, D114R, D114C, S134P, S134A , S134V, S134W, S134D, S134H, S134L, S134G, L137W, L137S, L137A, L137V, L1 37G, L137D, L137R, L137P, T139D, T139P, T139V, N142Y, N142H, N142C, L145 C, L145D, L145G, L145V, L145A, L145S, S146W, S146L, S146R, S146G, S146P, N 147Q, N147V, N147L, N147K, L147D, L147Y, L147H, L147S, N149H, N149T, N14 9R, N149K, N149S, Y152S, Y152A, Y152R, Y152L, Y152K, Y152E, Y152P, Y152V,Y152I、Y152C、Y152W、V153E、V153S、V153G、V153W、V153Y、T154N、T154R、T154K、T154P、T154V、S155C、S155P、S155R、S155G、S155A、N156I、N156A、N156V、N156R、N156T、N156K、L157P、L157R、L157A、L157G、L157W、W158T、W158A、W158M、W158V、W158R、W158P、P159S、P159G、P159L、P159V、P159A、P159R、P159Q、P159E、I160A、I160G、I160N、I160T、I160R、I160V、Q162L、Q162V、Q162H、Q162P、Q162R、V169A、V169L、V169W、V169S、V169D、V169R、V169E、S170A、S170P、S170R、S170M、S175L、S175C、S175W、T176R、T176L、T176N、T176A、T176S、T176I、Y177H、D184P、D184W、D184S、D184Y、D184G、S186A、S186R、S186W、R199F、R199E、R199L、R199C、R199K、A202R、A202W、A202E、A202S、A202V、A203M、A203W、A203P、A203L、T206C、T206P、T206G、T206A、T206R、Q210C、Q210G、Q210S、Q210R、Q210L、Q210P、Q210V、T211R、T211A、T211H、T211K、T211Q、T211G、T211W、T211E、T211I、T211V、T211P、T211L、T211D、S212D、S212E、S212L、S212P、S212T、Q213W、Q213V、Q213D、Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、F227G、F227W、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、S235C、S235R、S235N、S235G、S235W、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、T243V、T243S、T243L、T243R、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、R247E、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W、S248K、S248T、A252E、A252T、A252Y、A252V、A252L、T254D、T254W、T254V、T254G、T254A、L255R、L255Q、L255P、L255G、Y262C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A270L、A270M、A271V、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R、S296F、S296L、S296W、S296K、I297S、I297P、I297K、I297F、I297R、I297W、N298W、N298G、N298C、N298V、N298L、N298A、S299P、S299C、S299M、S299L、S299T、G300S, G300A, G300P, G300L, G300W, A302G, A302L, A302C, A302R, A302V, S303P, S303V, S303C, S303A, S303R, N304T, N304R, N304Q, N304L, N304V, T309 G、T309I、T309R、T309M、E314Y、E314T、E314V、E314G、E314S、E314L、E314A、 S316T、S316L、S316G、S316F、S316R、S316P、S316V、S316Q、Q318L、Q318R、G31 9R, G319Q, G319P, G319A, T326V, T326G, T326W, T326N, T326A, V330S, V330L, V330P, V330R, V330A, V330G, N339P, N339A, N339T, E342M, E342W, E342N, E 342L, E342R, S343R, S343C, E348W, E348F, E348P, E348V, E348G, E348M, S351P, S351C, S351G, S351R, S351L, S351W, T352P, T352L, T352G, T352Q, T352Y Q359K, Q359P, Q359R, Q359S, Q359A, S362P, S262R, S262G, S262M, G363R, G363T, G363P, V364A, V364C, V364E, V364S, V364G, V364L, T365S, T365G, T365 W, T365L, T365H, A366D, A366T, A366P, A366R, A366H, S371A, S371G, S372A, S372E, S372C, S372L, S372R, T378G, T378L, T378D, T378H, T378A, T378P, S38 1K、I383A、I383G、I383C、I383L、I383T、I383M、N385R、N385W、N385S、N385G 、N385D、F386S、F386W、F386Q、F386V、F386I、F386G、F386C、F386A、F386T、F 386L, A392V, A392L, A392E, A392G, N394D, N394R, N394Y, N394W, N394E, K396I, K396W, K396P, K396Y, K396F, Y408V, Y408E, Y408P, Y408S, Y408K, Y408LK410S、K410R、D412M、D412S、D412N、D412W、D412L、D412R、S414C、S414R、S414G、S414V、S414W、S414H、S417Y、V419S、V419G、V419C、V419A、V419K、V419R、V419T、A426M、A426N、A426K、A426R、S427G、S427A、S427P、S427N、S427D、S427L、E433C、A434Q、A434G、N436S、N436P、N436D、N437K、N437R、N437T、N437P、T438E、T438G、Q439W、Q439S、Q439G、Q439C、Q439R、Q439Y、G442V、G442D、G442C、G442A、G442L、G442W、G442E、G442M、G442R、A446G、A446D、A446R、A446E、A446I、L448G、L448P、L448E、V450P、V450S、V450C、V450E、V450L、V450N、N470H、N470D、N470K、N470V、N470L、E472I、V474W、V474C、V474A、V474L、V474G、W475P、W475A、W475R、N478L、N478I、N478P、N478R、N478W、N478S、N478G、N478K、N478A、S484G、S484Y、S484P、S484A、S484N、V485A、V485W、V485K、V485G、V485R、D486I、D486K、D486Y、D486S、D486A、D486W、D486L、A487S、A487V、A487L、A487G、A487C、A487K、S492L、S492R、S492T、S492W、S492P、S492C、A493V、A493R、A493D、A493W、D494N、D494R、D494G、D494L、D494E、D494Q、N495L、N495W、N495G、N495R、N495C、S501R、S501L、S501M、S501K、S501W、A502C、A502Q、A502W、A502G、A502V、T506A、T506P、T506V、I509E、I509D、I509S、I509F、I509W、I509R、T510F、T510E、T510R、T510P、T510V、T510A、T510L、N512Q, N512K, N512H, N512R, N512V, S516R, S516W, S516P, S516K, S516Y, S516C, A518D, A518 G, A518Y, A518V, A518R, A518L, A518T, I519W, I519L, I519R, I519F, I519K, N527T, N527K, N52 7P, N527L, N528D, N528G, N528K, N528V, N528E, N528L, A530R, A530C, A530G, A530V, A530S, A 530T, E534W, E534Q, E534C, E534V, E534G, E534R, E534F, E534K, D536G, D536R, D536W, D536H, D536K, D536N, D536M, D536C, D536V, P537D, P537M, P537W, P537G, P537E, N538D, N538S, N538 W, N538Y, N538A, N539M, N539R, N539P, N539A, I541A, I541T, I541V, I541G, I541N, A545R, A54 5T, A545V, A545L, S546P, S546G, S546C, S546E, S546N, G547D, G547S, G547V, S548P, S548W, S548L, S548G, S548T, N552V, N552E, N552F, N552A, N552R, N552G and T554Q, T554G, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in melting temperature of at least 0.5 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0060] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: S2, V3, D4, S8, S9, I13, V18, L19, S27, K28, S30, V36, V37, T43, S57, V59, S73, T74, L77, D82, V85, T86, L91, S95, P97, D114, S134, L137, T139, N142, L145, S146, N147, N149, Y152, V153, T154, S155, N156, L157. W158, P159, I160, Q162, S170, S175, T176, Y177, D184, S186, R199, A203, T206, Q210, T211, S212, Q213, V214, S215, Y217, T218, T219, Q 220, A221, D222, N223, L224, F227, Y231, P234, S235, Y238, T240, G244, G245, G246, S248, A252, T254, L255, Y262, S265, G267, A271, K27 9. S282, L284, V294, Y295, S296, I297, N298, S299, G300, S303, N304, T309, E314, S316, Q318, G319, T326, V330, N339, E342, S343, E348 , T352, Q359, G363, V364, A366, S371, S372, T378, S381, I383, N385, F386, A392, N394, K396, Y408, K410, D412, S414, S417, V419, A426, S427, A434, N436, T438, Q439, G442, L448, V450, N470, E472, V474, W475, S484, V485, D486, A487, S492, A493, D494, N495, A502, T506, I509, T510, N512, S516, A518, I519, N527, N528, A530, E534, P537, N538, N539, I541, A545, S546, G547, S548, N552, and T554, wherein the positions correspond to in SEQ The amino acid positions in the amino acid sequence shown in ID NO: 3; and the substitutions at one or more of the positions provided a glucosylamylase variant having at least 0 as measured by TSA.The melting temperature increases by 7 degrees Celsius, and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence similarity to the amino acid sequence having SEQ ID NO: 3.

[0061] In specific embodiments, the glucosylamylase variant comprises substitutions at one or more positions selected from the group consisting of: S2E, S2K, S2L, S2P, S2R, V3L, V3G, V3R, D4R, D4S, D4G, D4A, D4W, S8Q, S8H, S8A, S8Y, S9C, S9Q, S9M, S9W, S9D, S9G, I13V, I13R, I13S, I13L, I13E, V18M, V18Q, L19G, L19F, S27A, S27L, S27G, S27V, S27C, K28C, K28R, S30Q, S30A, S30K, S30T, S30L, V36K, V 36G, V36W, V36A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43 K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, V59G, V59T, V59S, V59E, S73H, S 73A, S73R, S73N, S73V, S73G, T74V, L77S, L77P, L77R, D82N, D82R, D82V, D82 G, V85Q, V85G, V85P, T86R, T86V, L91S, L91P, L91G, S95A, S95P, S95T, S95V, P 97T, P97I, P97R, D114G, D114N, D114M, D114R, D114C, S134P, S134A, S134V, S134W, S134D, S134H, S134L, S134G, L137W, L137S, L137A, L137V, L137G, L13 7D, L137R, L137P, T139D, T139P, T139V, N142Y, N142H, N142C, L145C, L145D , L145G, L145V, L145A, L145S, S146W, S146L, S146R, S146G, S146P, N147Q, N1 47V, N147L, N147K, L147D, L147Y, L147H, L147S, N149H, N149T, N149R, N149 K, N149S, Y152S, Y152A, Y152R, Y152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y 152C, Y152W, V153E, V153S, V153G, V153W, V153Y, T154N, T154R, T154K, T15 4P, T154V, S155C, S155P, S155R, S155G, S155A, N156I, N156A, N156V, N156R,N156T、N156K、L157P、L157R、L157A、L157G、L157W、W158T、W158A、W158M、W158V、W158R、W158P、P159S、P159G、P159L、P159V、P159A、P159R、P159Q、P159E、I160A、I160G、I160N、I160T、I160R、I160V、Q162L、Q162V、Q162H、Q162P、Q162R、S170A、S170P、S170R、S170M、S175L、S175C、S175W、T176R、T176L、T176N、T176A、T176S、T176I、Y177H、D184P、D184W、D184S、D184Y、D184G、S186A、S186R、S186W、R199F、R199E、R199L、R199C、R199K、A203M、A203W、A203P、A203L、T206C、T206P、T206G、T206A、T206R、Q210C、Q210G、Q210S、Q210R、Q210L、Q210P、Q210V、T211R、T211A、T211H、T211K、T211Q、T211G、T211W、T211E、T211I、T211V、T211P、T211L、T211D、S212D、S212E、S212L、S212P、S212T、Q213W、Q213V、Q213D、Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、F227G、F227W、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、S235C、S235R、S235N、S235G、S235W、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W、S248K、S248T、A252E、A252T、A252Y、A252V、A252L、T254D、T254W、T254V、T254G、T254A、L255R、L255Q、L255P、L255G、Y262C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A271V、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R、S296F、S296L、S296W、S296K、I297S、I297P、I297K、I297F、I297R、I297W、N298W、N298G、N298C、N298V、N298L、N298A、S299P、S299C、S299M、S299L、S299T、G300S、G300A、G300P、G300L、G300W、S303P、S303V、S303C、S303A、S303R、N304T、N304R、N304Q、N304L、N304V、T309G、T309I、T309R、T309M、E314Y、E314T、E314V、E314G、E314S、E314L、E314A、S316T、S316L、S316G、S316F、S316R、S316P、S316V、S316Q、Q318L、Q318R、G319R、G319Q、G319P、G319A、T326V、T326G, T326W, T326N, T326A, V330S, V330L, V330P, V330R, V330A, V330G, N339P, N339A, N339T, E342M, E342W, E342N, E342L, E342R, S343R, S343C, E348 W, E348F, E348P, E348V, E348G, E348M, T352P, T352L, T352G, T352Q, T352Y, Q359K, Q359P, Q359R, Q359S, Q359A, G363R, G363T, G363P, V364A, V364C, V36 4E、V364S、V364G、V364L、A366D、A366T、A366P、A366R、A366H、S371A、S371G 、S372A、S372E、S372C、S372L、S372R、T378G、T378L、T378D、T378H、T378A、T 378P, S381K, I383A, I383G, I383C, I383L, I383T, I383M, N385R, N385W, N385S, N385G, N385D, F386S, F386W, F386Q, F386V, F386I, F386G, F386C, F386A F386T, F386L, A392V, A392L, A392E, A392G, N394D, N394R, N394Y, N394W, N394E, K396I, K396W, K396P, K396Y, K396F, Y408V, Y408E, Y408P, Y408S, Y408 K、Y408L、K410S、K410R、D412M、D412S、D412N、D412W、D412L、D412R、S414C、 S414R、S414G、S414V、S414W、S414H、S417Y、V419S、V419G、V419C、V419A、V41 9K, V419R, V419T, A426M, A426N, A426K, A426R, S427G, S427A, S427P, S427N, S427D, S427L, A434Q, A434G, N436S, N436P, N436D, T438E, T438G, Q439W, Q 439S、Q439G、Q439C、Q439R、Q439Y、G442V、G442D、G442C、G442A、G442L、G44 2W、G442E、G442M、G442R、L448G、L448P、L448E、V450P、V450S、V450C、V450E、V450L、V450N、N470H、N470D、N470K、N470V、N470L、E472I、V474W、V474C、V474A、V474L、V474G、W475P、W475A、W475R、S484G、S484Y、S484P、S484A、S484N、V485A、V485W、V485K、V485G、V485R、D486I、D486K、D486Y、D486S、D486A、D486W、D486L、A487S、A487V、A487L、A487G、A487C、A487K、S492L、S492R、S492T、S492W、S492P、S492C、A493V、A493R、A493D、A493W、D494N、D494R、D494G、D494L、D494E、D494Q、N495L、N495W、N495G、N495R、N495C、A502C、A502Q、A502W、A502G、A502V、T506A、T506P、T506V、I509E、I509D、I509S、I509F、I509W、I509R、T510F、T510E、T510R、T510P、T510V、T510A、T510L、N512Q、N512K、N512H、N512R、N512V、S516R、S516W、S516P、S516K、S516Y、S516C、A518D、A518G、A518Y、A518V、A518R、A518L、A518T、I519W、I519L、I519R、I519F、I519K、N527T、N527K、N527P、N527L、N528D、N528G、N528K、N528V、N528E、N528L、A530R、A530C、A530G、A530V、A530S、A530T、E534W、E534Q、E534C、E534V、E534G、E534R、E534F、E534K、P537D、P537M、P537W、P537G、P537E、N538D、N538S、N538W、N538Y、N538A、N539M、N539R、N539P、N539A、I541A、I541T、I541V、I541G、I541N、A545R、A545T、A545V、A545L、S546P、S546G、S546C、S546E、S546N、G547D、G547S、G547V、S548P、S548W、S548L、S548G、S548T、N552V、N552E、N552F, N552A, N552R, N552G, T554Q, and T554G, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in melting temperature of at least 0.7 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0062] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: S2, S8, V18, K28, V36, V37, T43, S57, V59, S73, T74, T86, S95, P97, D114, S134, L137, T139, N142, L145, S146, N147, N149, Y152, V153, T154, S155 , L157, W158, P159, I160, Q162, S170, S175, T176, Y177, D184, S186, R199, A203, T206, Q210, T211, S212 , Q213, V214, S215, Y217, T218, T219, Q220, A221, D222, N223, L224, F227, Y231, P234, Y238, T240, G244, G245, G246, S248, A252, T254, L255, Y262, S265, G267, A271, K279, S282, L284, V294, Y295, S296, I297, N298, G300, S303, N304, T309, E314, Q318, T326, V330, E342, S343, E348, T352, Q359, S371, T378, I383, F 386, N394, K396, Y408, K410, D412, S417, V419, A426, S427, A434, Q439, G442, L448, N470, E472, W475, V485, D486, S492, A493, D494, N495, I509, T510, I519, N527, P537, N538, S546, G547, and T554, wherein the positions correspond to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitution at one or more of the positions provides a glucosylamylase variant having an increase in melting temperature of at least 0.9 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0063] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: S2E, S2K, S2L, S2P, S2R, S8Q, S8H, S8A, S8Y, V18M, V18Q, K28C, K28R, V36K, V36G, V36W, V36A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, V59G, V59T, V59S, V59E, S73H, S73A, S73R. S73N, S73V, S73G, T74V, T86R, T86V, S95A, S95P, S95T, S95V, P97T, P97I, P9 7R, D114G, D114N, D114M, D114R, D114C, S134P, S134A, S134V, S134W, S134D, S134H, S134L, S134G, L137W, L137S, L137A, L137V, L137G, L137D, L137R, L1 37P, T139D, T139P, T139V, N142Y, N142H, N142C, L145C, L145D, L145G, L145V , L145A, L145S, S146W, S146L, S146R, S146G, S146P, N147Q, N147V, N147L, N 147K, L147D, L147Y, L147H, L147S, N149H, N149T, N149R, N149K, N149S, Y15 2S, Y152A, Y152R, Y152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C, Y152W , V153E, V153S, V153G, V153W, V153Y, T154N, T154R, T154K, T154P, T154V, S1 55C, S155P, S155R, S155G, S155A, L157P, L157R, L157A, L157G, L157W, W158 T, W158A, W158M, W158V, W158R, W158P, P159S, P159G, P159L, P159V, P159A, P 159R, P159Q, P159E, I160A, I160G, I160N, I160T, I160R, I160V, Q162L, Q16 2V, Q162H, Q162P, Q162R, S170A, S170P, S170R, S170M, S175L, S175C, S175W,T176R、T176L、T176N、T176A、T176S、T176I、Y177H、D184P、D184W、D184S、D184Y、D184G、S186A、S186R、S186W、R199F、R199E、R199L、R199C、R199K、A203M、A203W、A203P、A203L、T206C、T206P、T206G、T206A、T206R、Q210C、Q210G、Q210S、Q210R、Q210L、Q210P、Q210V、T211R、T211A、T211H、T211K、T211Q、T211G、T211W、T211E、T211I、T211V、T211P、T211L、T211D、S212D、S212E、S212L、S212P、S212T、Q213W、Q213V、Q213D、Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、F227G、F227W、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、S248Y、S248P、S248V、S248L、S248F, S248A, S248E, S248W, S248K, S248T, A252E, A252T, A252Y, A252V, A252L, T254D, T254W, T254V, T254G, T254A, L255R, L255Q, L255P, L255G, Y262 C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、 G267W、G267C、A271V、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K27 9G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R 、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R、S296F、S296L、S 296W、S296K、I297S、I297P、I297K、I297F、I297R、I297W、N298W、N298G、N29 8C、N298V、N298L、N298A、G300S、G300A、G300P、G300L、G300W、S303P、S303V、 S303C, S303A, S303R, N304T, N304R, N304Q, N304L, N304V, T309G, T309I, T309R, T309M, E314Y, E314T, E314V, E314G, E314S, E314L, E314A, Q318L, Q318 R、T326V、T326G、T326W、T326N、T326A、V330S、V330L、V330P、V330R、V330A、 V330G、E342M、E342W、E342N、E342L、E342R、S343R、S343C、E348W、E348F、E34 8P、E348V、E348G、E348M、T352P、T352L、T352G、T352Q、T352Y、Q359K、Q359P 、Q359R、Q359S、Q359A、S371A、S371G、T378G、T378L、T378D、T378H、T378A、T 378P, I383A, I383G, I383C, I383L, I383T, I383M, F386S, F386W, F386Q, F386V, F386I, F386G, F386C, F386A, F386T, F386L, N394D, N394R, N394Y, N394WN394E, K396I, K396W, K396P, K396Y, K396F, Y408V, Y408E, Y408P, Y408S, Y408K, Y408L, K410S, K410R, D412M, D412S, D412N, D412W, D412L, D412R, S417Y, V419S, V419G, V419C, V419A, V419K, V419R, V419T, A426M, A426N, A426K, A426R, S427G, S427A, S427P, S427N, S427D, S427L, A434Q, A434G, Q439W, Q439S, Q439G, Q439C, Q439R, Q439Y, G442V, G442D, G442C, G442A, G442L, G442W, G442E, G442M, G442R, L448G, L448P, L448E, N470H, N470D, N470K, N470V, N470L, E472I, W475P, W475A, W475R, V485A, V485W, V485K, V485G, V485R, D486I, D486K, D486Y, D486S, D486A, D486W, D486L, S492L, S492R, S492T, S492W, S492P, S492C, A493V, A493R, A493D, A493W, D494N, D494R, D494G, D494L, D494E, D494Q, N495L, N495W, N495G, N495R, N495C, I509E, I509D, I509S, I509F, I509W, I509R, T510F, T510E, T510R, T510P, T510V, T510A, T510L, I519W, I519L, I519R, I519F, I519K, N527T, N527K, N527P, N527L, P537D, P537M, P537W, P537G, P537E, N538D, N538S, N538W, N538Y, N538A, S546P, S546G, S546C, S546E, S546N, G547D, G547S, G547V, T554Q, and T554G, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more of the positions provide a glucosylamylase variant having an increase in melting temperature of at least 0.9 degrees Celsius as measured by TSA, and further wherein the variant has at least 85% and at least 90% amino acid content with the amino acid sequence having SEQ ID NO: 3.At least 95% sequence consistency, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, of sequence consistency.

[0064] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: V18, K28, V36, V37, T43, S57, V59, S73, T86, S95, T139, N142, L145, S146, N147, N149, Y152, V153, T154, L157, W158, P159, S170, S175, T176, D184, S186, R199, A203, Q210, T211, Q213, V214, S215, Y217, T218, T219, Q220, A221, D222, N223, L224, Y231, P234, Y238, T 240, G244, G245, G246, S248, A252, T254, L255, Y262, S265, G267, A271, K279, S282 , L284, V294, Y295, S296, I297, N298, G300, S303, N304, T309, E314, T326, E342, S3 43, T352, Q359, I383, K396, K410, D412, S417, V419, A426, S427, A434, Q439, N470, W475, D486, S492, A493, D494, N495, T510, P537, N538, G547, and T554, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitution at one or more positions provides a glucosylamylase variant having an increase in melting temperature of at least 1.1 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0065] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: V18M, V18Q, K28C, K28R, V36K, V36G, V36W, V36A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, V59G, V59T, V59S, V59E, S73H, S73A, S73R, S73N, S73V, S73G, T86R, T86V, S95A, S95P, S 95T, S95V, T139D, T139P, T139V, N142Y, N142H, N142C, L145C, L145D, L145G , L145V, L145A, L145S, S146W, S146L, S146R, S146G, S146P, N147Q, N147V, N 147L, N147K, L147D, L147Y, L147H, L147S, N149H, N149T, N149R, N149K, N14 9S, Y152S, Y152A, Y152R, Y152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C, Y152W, V153E, V153S, V153G, V153W, V153Y, T154N, T154R, T154K, T154P, T1 54V, L157P, L157R, L157A, L157G, L157W, W158T, W158A, W158M, W158V, W158 R, W158P, P159S, P159G, P159L, P159V, P159A, P159R, P159Q, P159E, S170A, S170P, S170R, S170M, S175L, S175C, S175W, T176R, T176L, T176N, T176A, T17 6S, T176I, D184P, D184W, D184S, D184Y, D184G, S186A, S186R, S186W, R199F , R199E, R199L, R199C, R199K, A203M, A203W, A203P, A203L, Q210C, Q210G, Q 210S, Q210R, Q210L, Q210P, Q210V, T211R, T211A, T211H, T211K, T211Q, T21 1G, T211W, T211E, T211I, T211V, T211P, T211L, T211D, Q213W, Q213V, Q213D,Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W、S248K、S248T、A252E、A252T、A252Y、A252V、A252L、T254D、T254W、T254V、T254G、T254A、L255R、L255Q、L255P、L255G、Y262C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A271V、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R, S296F, S296L, S296W, S296K, I297S, I297P, I297K, I297F, I297R, I297W, N298W, N298G, N298C, N298V, N298L, N298A, G300S, G300A, G300P, G300 L, G300W, S303P, S303V, S303C, S303A, S303R, N304T, N304R, N304Q, N304L, N304V, T309G, T309I, T309R, T309M, E314Y, E314T, E314V, E314G, E314S, E31 4L、E314A、T326V、T326G、T326W、T326N、T326A、E342M、E342W、E342N、E342L 、E342R、S343R、S343C、T352P、T352L、T352G、T352Q、T352Y、Q359K、Q359P、Q 359R, Q359S, Q359A, I383A, I383G, I383C, I383L, I383T, I383M, K396I, K396W, K396P, K396Y, K396F, K410S, K410R, D412M, D412S, D412N, D412W, D412L D412R、S417Y、V419S、V419G、V419C、V419A、V419K、V419R、V419T、A426M、A4 26N、A426K、A426R、S427G、S427A、S427P、S427N、S427D、S427L、A434Q、A434 G、Q439W、Q439S、Q439G、Q439C、Q439R、Q439Y、N470H、N470D、N470K、N470V、 N470L、W475P、W475A、W475R、D486I、D486K、D486Y、D486S、D486A、D486W、D48 6L、S492L、S492R、S492T、S492W、S492P、S492C、A493V、A493R、A493D、A493W 、D494N、D494R、D494G、D494L、D494E、D494Q、N495L、N495W、N495G、N495R、N 495C、T510F、T510E、T510R、T510P、T510V、T510A、T510L、P537D、P537M、P53 7W、P537G、P537E、N538D、N538S、N538W、N538Y、N538A、G547D、G547S、G547V、T554Q and T554G, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in melting temperature of at least 1.1 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0066] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: K28, T43, S57, S73, S95, T139, L145, N149, Y152, V153, L157, W158, P159, S175, D184, S186, R199, A203, Q210, T211, Q213, V214, Y217, T218, T219, A221. D222, L224, Y231, P234, Y238, T240, G244, G246, S248, A252, T254, L255, Y262, A271, K279, L284, V294, Y295, S296, I297, N298, N304, S343, T352, K410, D412, V419, S427, N470, W475, S492, A493, and P537, wherein the positions correspond to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitution at one or more of the positions provides a glucosylamylase variant having an increase in melting temperature of at least 1.5 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0067] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: K28C, K28R, T43K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, S73H, S73A, S73R, S73N, S73V, S73G, S95A, S95P, S95T, S95V, T139D, T139P, T139V, L145C, L145D, L145G, L145V, L145A, L145S, N149H, N149T, N149R, N149K, N149S, Y152S, Y152A. Y152R, Y152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C, Y152W, V153E, V1 53S, V153G, V153W, V153Y, L157P, L157R, L157A, L157G, L157W, W158T, W158A , W158M, W158V, W158R, W158P, P159S, P159G, P159L, P159V, P159A, P159R, P 159Q, P159E, S175L, S175C, S175W, D184P, D184W, D184S, D184Y, D184G, S186 A. S186R, S186W, R199F, R199E, R199L, R199C, R199K, A203M, A203W, A203P, A203L, Q210C, Q210G, Q210S, Q210R, Q210L, Q210P, Q210V, T211R, T211A, T21 1H, T211K, T211Q, T211G, T211W, T211E, T211I, T211V, T211P, T211L, T211D , Q213W, Q213V, Q213D, Q213A, Q213T, Q213R, Q213G, Q213S, V214G, V214R, V2 14W, V214A, V214I, Y217G, Y217C, Y217A, Y217S, Y217T, Y217F, T218H, T218 C. T218A, T218M, T218Q, T218G, T219R, T219D, T219S, T219G, T219C, A221V, A 221T, A221L, A221P, A221R, A221E, D222V, D222W, D222T, D222G, D222L, D22 2R, D222N, D222F, D222M, L224G, L224D, L224K, L224V, L224R, Y231S, Y231T,Y231R, Y231L, Y231A, Y231V, Y231N, P234D, P234L, P234S, P234V, Y238R, Y238A, Y238Q, Y238C, Y238E, T240C, T240I, T240L, T240S, G244R, G2 44C, G244P, G244D, G244W, G246L, G246E, G246S, G246R, G246K, G246W, G246D, S248Y, S248P, S248V, S248L, S248F, S248A, S248E, S248W, S248 K, S248T, A252E, A252T, A252Y, A252V, A252L, T254D, T254W, T254V, T254G, T254A, L255R, L255Q, L255P, L255G, Y262C, Y262Q, Y262S, Y262G, Y262V, Y262A, Y262W, A271V, A271W, A271Y, A271L, K279R, K279W, K279 E. K279P, K279G, K279F, L284N, L284Q, L284T, L284S, L284R, L284G, L28 4V, V294G, V294W, V294E, V294S, Y295V, Y295R, S296F, S296L, S296W, S 296K, I297S, I297P, I297K, I297F, I297R, I297W, N298W, N298G, N298C , N298V, N298L, N298A, N304T, N304R, N304Q, N304L, N304V, S343R, S34 3C, T352P, T352L, T352G, T352Q, T352Y, K410S, K410R, D412M, D412S, D 412N, D412W, D412L, D412R, V419S, V419G, V419C, V419A, V419K, V419R, V419T, S427G, S427A, S427P, S427N, S427D, S427L, N470H, N470D, N470K, N470V, N470L, W475P, W475A, W475R, S492L, S492R, S492T, S492W, S492P, S492C, A493V, A493R, A493D, A493W, and P537D, P537M, P537W, P537G.P537E, wherein the position corresponds to an amino acid position in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitution at one or more positions provides a glucosylamylase variant having an increase in melting temperature of at least 1.5 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0068] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions selected from the group consisting of: K28, T43, S57, S95, T139, L145, N149, V153, L157, P159, S186, R199, A203, T211, V214, Y217, Y231, P234, Y238, T240, G244, G246, S248, T254, L255, A271, L284, V294, Y295, S296, K410, S492, and A493, wherein the positions correspond to SEQ ID. The amino acid positions in the amino acid sequence shown in NO:3; and the substitutions at one or more of the positions provide a glucosylamylase variant having an increase in melting temperature of at least 2.0 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence having SEQ ID NO:3.

[0069] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group below.This group consists of the following: K28C, K28R, T43K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, S95A, S95P, S95T, S95V, T139D, T139P, T139V, L145C, L145D, L145G, L145V, L145A, L145S, N149H, N149T, N149R, N149K, N149S, V153E, V153S, V153G, V153W, V153Y, L157P, L157R, L157A, L157G, L157W, P159S, P159G, P159L, P159V. P159A, P159R, P159Q, P159E, S186A, S186R, S186W, R199F, R199E, R199L, R1 99C, R199K, A203M, A203W, A203P, A203L, T211R, T211A, T211H, T211K, T211 Q. T211G, T211W, T211E, T211I, T211V, T211P, T211L, T211D, V214G, V214R, V214W, V214A, V214I, Y217G, Y217C, Y217A, Y217S, Y217T, Y217F, Y231S, Y2 31T, Y231R, Y231L, Y231A, Y231V, Y231N, P234D, P234L, P234S, P234V, Y238 R, Y238A, Y238Q, Y238C, Y238E, T240C, T240I, T240L, T240S, G244R, G244C, G244P, G244D, G244W, G246L, G246E, G246S, G246R, G246K, G246W, G246D, S2 48Y, S248P, S248V, S248L, S248F, S248A, S248E, S248W, S248K, S248T, T254 D. T254W, T254V, T254G, T254A, L255R, L255Q, L255P, L255G, A271V, A271W, A271Y, A271L, L284N, L284Q, L284T, L284S, L284R, L284G, L284V, V294G, V2 94W, V294E, V294S, Y295V, Y295R, S296F, S296L, S296W, S296K, K410S, K410R, S492L, S492R, S492T, S492W, S492P, S492C, A493V, A493R, A493D, and A493W,The positions described therein correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more positions provide a glucosylamylase variant having an increase in melting temperature of at least 2.0 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0070] In specific embodiments, the glucosylamylase variant comprises substitutions at one or more positions selected from the group consisting of: Q1K, Q1R, S2E, S2K, S2L, S2P, S2R, V3L, V3G, V3R, D4R, D4S, D4G, D4A, D4W, S5L, S5V, S5G, S5C, S5R, S8Q, S8H, S8A, S8Y, S9C, S9Q, S9M, S9W, S9D, S9G, I13V, I13R, I13S, I13L, I13E, K15G, K15R, V18M, V18Q, L19G, L19F, N25S, N25A, S27A, S27L, S27G, S27V , S27C, K28C, K28R, S30Q, S30A, S30K, S30T, S30L, V36K, V36G, V36W, V36A, V 36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, D45L, D45P, S57P , S57L, S57G, S57F, S57R, S57T, S57A, V59T, V59S, V59E, F60S, I71M, I71S, I 71T, I71V, S73H, S73R, S73N, S73V, S73G, T74V, L77S, L77P, L77R, D82N, D82R , D82V, D82G, D83L, D83C, D83W, V85Q, V85G, V85P, T86R, T86V, E88Q, E88R, E 88G, L91S, L91P, L91G, S95A, S95T, S95V, P97T, P97I, P97R, T103Y, T103A, T1 03G, D114G, D114N, D114M, D114R, D114C, S134P, S134A, S134V, S134W, S134 D. S134H, S134L, S134G, L137W, L137S, L137A, L137V, L137G, L137D, L137R, L 137P, T139D, T139P, T139V, N142Y, N142H, N142C, L145C, L145D, L145G, L14 5V, L145S, S146W, S146L, S146R, S146G, S146P, N147Q, N147V, N147L, N147K, L147D, L147Y, L147H, L147S, N149H, N149T, N149R, N149K, N149S, Y152S, Y15 2A, Y152R, Y152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C, Y152W, V153E,V153S、V153G、V153W、V153Y、T154N、T154R、T154K、T154P、T154V、S155C、S155P、S155R、S155G、S155A、N156I、N156A、N156V、N156R、N156T、N156K、L157P、L157R、L157A、L157G、L157W、W158T、W158A、W158M、W158V、W158R、W158P、P159S、P159G、P159L、P159V、P159A、P159R、P159Q、P159E、I160A、I160G、I160N、I160T、I160R、I160V、Q162L、Q162V、Q162H、Q162P、Q162R、V169A、V169L、V169W、V169S、V169D、V169R、V169E、S170A、S170P、S170R、S170M、S175L、S175C、S175W、T176R、T176L、T176N、T176A、T176S、T176I、Y177H、D184P、D184W、D184S、D184Y、D184G、S186A、S186R、S186W、R199F、R199E、R199L、R199C、R199K、A202R、A202W、A202E、A202S、A202V、A203M、A203W、A203P、A203L、T206C、T206P、T206G、T206A、T206R、Q210C、Q210G、Q210S、Q210R、Q210L、Q210P、Q210V、T211R、T211A、T211H、T211K、T211Q、T211G、T211W、T211E、T211I、T211V、T211P、T211L、T211D、S212D、S212E、S212L、S212P、S212T、Q213W、Q213V、Q213D、Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、F227G、F227W、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、S235C、S235R、S235N、S235G、S235W、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、T243V、T243S、T243L、T243R、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、R247E、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W、S248K、S248T、A252E、A252T、A252Y、A252V、A252L、T254D、T254W、T254V、T254G、T254A、L255R、L255Q、L255P、L255G、Y262C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A270L、A270M、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R、S296F、S296L、S296W、S296K、I297S、I297P、I297K、I297F、I297R、I297W、N298W、N298G、N298C、N298V、N298L、N298A、S299P、S299C、S299M、S299L、S299T、G300S、G300A、G300P、G300L、G300W、A302G, A302L, A302C, A302R, A302V, S303P, S303V, S303C, S303A, S303R, N304T, N304R, N304Q, N304L, N304V, T309G, T309I, T309R, T309M, E314Y, E314 T、E314V、E314G、E314S、E314L、E314A、S316T、S316L、S316G、S316F、S316R、 S316P、S316V、S316Q、Q318L、Q318R、G319R、G319Q、G319P、G319A、T326V、T32 6G、T326W、T326N、T326A、V330S、V330L、V330P、V330R、V330A、V330G、N339P 、N339A、N339T、E342M、E342W、E342N、E342L、E342R、S343R、S343C、E348W、E 348F, E348P, E348V, E348G, E348M, S351P, S351C, S351G, S351R, S351L, S351W, T352P, T352L, T352G, T352Q, T352Y, Q359K, Q359P, Q359R, Q359S, Q359A S362P, S262R, S262G, S262M, G363R, G363T, G363P, V364A, V364C, V364E, V364S, V364G, V364L, T365S, T365G, T365W, T365L, T365H, A366D, A366T, A366 P, A366R, A366H, S371A, S371G, S372A, S372E, S372C, S372L, S372R, T378G, T378L, T378D, T378H, T378A, T378P, S381K, I383A, I383G, I383C, I383L, I38 3T、I383M、N385R、N385W、N385S、N385G、N385D、F386S、F386W、F386Q、F386V 、F386I、F386G、F386C、F386A、F386T、F386L、A392V、A392L、A392E、A392G、N 394D、N394R、N394Y、N394W、N394E、K396I、K396W、K396P、K396Y、K396F、Y40 8V、Y408E、Y408P、Y408S、Y408K、Y408L、K410S、K410R、D412M、D412S、D412N、D412W、D412L、D412R、S414C、S414R、S414G、S414V、S414W、S414H、S417Y、V419S、V419G、V419C、V419A、V419K、V419R、V419T、A426M、A426N、A426K、A426R、S427G、S427A、S427P、S427N、S427D、S427L、E433C、A434Q、A434G、N436S、N436P、N436D、N437K、N437R、N437T、N437P、T438E、T438G、Q439W、Q439S、Q439G、Q439C、Q439R、Q439Y、G442V、G442D、G442C、G442A、G442L、G442W、G442E、G442M、G442R、A446G、A446D、A446R、A446E、A446I、L448G、L448P、L448E、V450P、V450S、V450C、V450E、V450L、V450N、N470H、N470D、N470K、N470V、N470L、E472I、V474W、V474C、V474A、V474L、V474G、W475P、W475A、W475R、N478L、N478I、N478P、N478R、N478W、N478S、N478G、N478K、N478A、S484G、S484Y、S484P、S484A、S484N、V485A、V485W、V485K、V485G、V485R、D486I、D486K、D486Y、D486S、D486A、D486W、D486L、A487S、A487V、A487L、A487G、A487C、A487K、S492L、S492R、S492T、S492W、S492P、S492C、A493V、A493R、A493D、A493W、D494N、D494R、D494G、D494L、D494E、D494Q、N495L、N495W、N495G、N495R、N495C、S501R、S501L、S501M、S501K、S501W、A502C、A502Q、A502W、A502G、A502V、T506A、T506P、T506V、I509E、I509D、I509S、I509F、I509W、I509R、T510F、T510E、T510R、T510P、T510V、T510A、T510L、N512Q、N512K、N512H、N512R、N512V、S516R, S516W, S516P, S516K, S516Y, S516C, A518D, A518G, A518Y, A518V, A518R, A518L, A518T, I519W, I519L, I519R, I519F, I519K, N527T, N527K, N527P, N527L, N528D, N528G, N 528K, N528V, N528E, N528L, A530R, A530C, A530G, A530V, A530S, A530T, E534W, E534Q, E5 34C, E534V, E534G, E534R, E534F, E534K, D536G, D536R, D536W, D536H, D536K, D536N, D53 6M, D536C, D536V, P537D, P537M, P537W, P537G, P537E, N538D, N538S, N538W, N538Y, N538 A. N539M, N539R, N539P, N539A, I541A, I541T, I541V, I541G, I541N, A545R, A545T, A545V The amino acids A545L, S546P, S546G, S546C, S546E, S546N, G547D, G547S, G547V, S548P, S548W, S548L, S548G, S548T, N552V, N552E, N552F, N552A, N552R, N552G, and T554Q, T554G, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitution at one or more positions provides a glucosylamylase variant having an increase in melting temperature of at least 0.5 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95% sequence identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, of the amino acid sequence having SEQ ID NO: 3. ,

[0071] In a specific embodiment, the glucosylamylase variant comprises substitutions at one or more positions selected from the group consisting of: S2E, S2K, S2L, S2P, S2R, V3L, V3G, V3R, D4R, D4S, D4G, D4A, D4W, S8Q, S8H, S8A, S8Y, S9C, S9Q, S9M, S9W, S9D, S9G, I13V, I13R, I13S, I13L, I13E, V18M, V18Q, L19G, L19F, S27A, S27L, S27G, S27V, S27C, K28C, K28R, S30Q, S30A, S30K, S30T, S30L, V36K, V36 G, V36W, V36A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, V59T, V59S, V59E, S73H, S73R, S73 N, S73V, S73G, T74V, L77S, L77P, L77R, D82N, D82R, D82V, D82G, V85Q, V85G, V85P, T86R, T86V, L91S, L91P, L91G, S95A, S95T, S95V, P97T, P97I, P97R, D11 4G, D114N, D114M, D114R, D114C, S134P, S134A, S134V, S134W, S134D, S134H , S134L, S134G, L137W, L137S, L137A, L137V, L137G, L137D, L137R, L137P, T1 39D, T139P, T139V, N142Y, N142H, N142C, L145C, L145D, L145G, L145V, L145 S, S146W, S146L, S146R, S146G, S146P, N147Q, N147V, N147L, N147K, L147D, L 147Y, L147H, L147S, N149H, N149T, N149R, N149K, N149S, Y152S, Y152A, Y15 2R, Y152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C, Y152W, V153E, V153S, V153G, V153W, V153Y, T154N, T154R, T154K, T154P, T154V, S155C, S155P, S15 5R, S155G, S155A, N156I, N156A, N156V, N156R, N156T, N156K, L157P, L157R,L157A、L157G、L157W、W158T、W158A、W158M、W158V、W158R、W158P、P159S、P159G、P159L、P159V、P159A、P159R、P159Q、P159E、I160A、I160G、I160N、I160T、I160R、I160V、Q162L、Q162V、Q162H、Q162P、Q162R、S170A、S170P、S170R、S170M、S175L、S175C、S175W、T176R、T176L、T176N、T176A、T176S、T176I、Y177H、D184P、D184W、D184S、D184Y、D184G、S186A、S186R、S186W、R199F、R199E、R199L、R199C、R199K、A203M、A203W、A203P、A203L、T206C、T206P、T206G、T206A、T206R、Q210C、Q210G、Q210S、Q210R、Q210L、Q210P、Q210V、T211R、T211A、T211H、T211K、T211Q、T211G、T211W、T211E、T211I、T211V、T211P、T211L、T211D、S212D、S212E、S212L、S212P、S212T、Q213W、Q213V、Q213D、Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、F227G、F227W、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、S235C、S235R、S235N、S235G、S235W、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W、S248K、S248T、A252E、A252T、A252Y、A252V、A252L、T254D、T254W、T254V、T254G、T254A、L255R、L255Q、L255P、L255G、Y262C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R、S296F、S296L、S296W、S296K、I297S、I297P、I297K、I297F、I297R、I297W、N298W、N298G、N298C、N298V、N298L、N298A、S299P、S299C、S299M、S299L、S299T、G300S、G300A、G300P、G300L、G300W、S303P、S303V、S303C、S303A、S303R、N304T、N304R、N304Q、N304L、N304V、T309G、T309I、T309R、T309M、E314Y、E314T、E314V、E314G、E314S、E314L、E314A、S316T、S316L、S316G、S316F、S316R、S316P、S316V、S316Q、Q318L、Q318R、G319R、G319Q、G319P、G319A、T326V、T326G、T326W、T326N、T326A、V330S、V330L, V330P, V330R, V330A, V330G, N339P, N339A, N339T, E342M, E342W, E342N, E342L, E342R, S343R, S343C, E348W, E348F, E348P, E348V, E348G, E348 M, T352P, T352L, T352G, T352Q, T352Y, Q359K, Q359P, Q359R, Q359S, Q359A, G363R, G363T, G363P, V364A, V364C, V364E, V364S, V364G, V364L, A366D, A36 6T、A366P、A366R、A366H、S371A、S371G、S372A、S372E、S372C、S372L、S372R 、T378G、T378L、T378D、T378H、T378A、T378P、S381K、I383A、I383G、I383C、I 383L, I383T, I383M, N385R, N385W, N385S, N385G, N385D, F386S, F386W, F386Q, F386V, F386I, F386G, F386C, F386A, F386T, F386L, A392V, A392L, A392E A392G、N394D、N394R、N394Y、N394W、N394E、K396I、K396W、K396P、K396Y、K3 96F、Y408V、Y408E、Y408P、Y408S、Y408K、Y408L、K410S、K410R、D412M、D412 S、D412N、D412W、D412L、D412R、S414C、S414R、S414G、S414V、S414W、S414H、 S417Y、V419S、V419G、V419C、V419A、V419K、V419R、V419T、A426M、A426N、A42 6K, A426R, S427G, S427A, S427P, S427N, S427D, S427L, A434Q, A434G, N436S, N436P, N436D, T438E, T438G, Q439W, Q439S, Q439G, Q439C, Q439R, Q439Y, G 442V、G442D、G442C、G442A、G442L、G442W、G442E、G442M、G442R、L448G、L44 8P、L448E、V450P、V450S、V450C、V450E、V450L、V450N、N470H、N470D、N470K、N470V、N470L、E472I、V474W、V474C、V474A、V474L、V474G、W475P、W475A、W475R、S484G、S484Y、S484P、S484A、S484N、V485A、V485W、V485K、V485G、V485R、D486I、D486K、D486Y、D486S、D486A、D486W、D486L、A487S、A487V、A487L、A487G、A487C、A487K、S492L、S492R、S492T、S492W、S492P、S492C、A493V、A493R、A493D、A493W、D494N、D494R、D494G、D494L、D494E、D494Q、N495L、N495W、N495G、N495R、N495C、A502C、A502Q、A502W、A502G、A502V、T506A、T506P、T506V、I509E、I509D、I509S、I509F、I509W、I509R、T510F、T510E、T510R、T510P、T510V、T510A、T510L、N512Q、N512K、N512H、N512R、N512V、S516R、S516W、S516P、S516K、S516Y、S516C、A518D、A518G、A518Y、A518V、A518R、A518L、A518T、I519W、I519L、I519R、I519F、I519K、N527T、N527K、N527P、N527L、N528D、N528G、N528K、N528V、N528E、N528L、A530R、A530C、A530G、A530V、A530S、A530T、E534W、E534Q、E534C、E534V、E534G、E534R、E534F、E534K、P537D、P537M、P537W、P537G、P537E、N538D、N538S、N538W、N538Y、N538A、N539M、N539R、N539P、N539A、I541A、I541T、I541V、I541G、I541N、A545R、A545T、A545V、A545L、S546P、S546G、S546C、S546E、S546N、G547D、G547S、G547V、S548P、S548W、S548L、S548G、S548T、N552V、N552E、N552F、N552A、N552R、N552G、T554Q and T554G, wherein the position corresponds to an amino acid position in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitution at one or more positions provides a glucosylamylase variant having an increase in melting temperature of at least 0.7 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0072] In another embodiment, the present invention relates to glucosylamylase variants comprising variants at one or more locations selected from the group consisting of: S2E, S2K, S2L, S2P, S2R, S8Q, S8H, S8A, S8Y, V18M, V18Q, K28C, K28R, V36K, V36G, V36W, V36A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, V59T, V59S, V59E, S73H, S73A, S73R, S73N, S 73V, S73G, T74V, T86R, T86V, S95A, S95T, S95V, P97T, P97I, P97R, D114G, D1 14N, D114M, D114R, D114C, S134P, S134A, S134V, S134W, S134D, S134H, S134L , S134G, L137W, L137S, L137A, L137V, L137G, L137D, L137R, L137P, T139D, T 139P, T139V, N142Y, N142H, N142C, L145C, L145D, L145G, L145V, L145S, S146 W, S146L, S146R, S146G, S146P, N147Q, N147V, N147L, N147K, L147D, L147Y, L147H, L147S, N149H, N149T, N149R, N149K, N149S, Y152S, Y152A, Y152R, Y15 2L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C, Y152W, V153E, V153S, V153G , V153W, V153Y, T154N, T154R, T154K, T154P, T154V, S155C, S155P, S155R, S1 55G, S155A, L157P, L157R, L157A, L157G, L157W, W158T, W158A, W158M, W158 V, W158R, W158P, P159S, P159G, P159L, P159V, P159A, P159R, P159Q, P159E, I 160A, I160G, I160N, I160T, I160R, I160V, Q162L, Q162V, Q162H, Q162P, Q16 2R, S170A, S170P, S170R, S170M, S175L, S175C, S175W, T176R, T176L, T176N,T176A、T176S、T176I、Y177H、D184P、D184W、D184S、D184Y、D184G、S186A、S186R、S186W、R199F、R199E、R199L、R199C、R199K、A203M、A203W、A203P、A203L、T206C、T206P、T206G、T206A、T206R、Q210C、Q210G、Q210S、Q210R、Q210L、Q210P、Q210V、T211R、T211A、T211H、T211K、T211Q、T211G、T211W、T211E、T211I、T211V、T211P、T211L、T211D、S212D、S212E、S212L、S212P、S212T、Q213W、Q213V、Q213D、Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、F227G、F227W、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W, S248K, S248T, A252E, A252T, A252Y, A252V, A252L, T254D, T254W, T254V, T254G, T254A, L255R, L255Q, L255P, L255G, Y262C, Y262Q, Y262S, Y262 G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A271W、 A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S28 2K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W 、V294E、V294S、Y295V、Y295R、S296F、S296L、S296W、S296K、I297S、I297P、I 297K、I297F、I297R、I297W、N298W、N298G、N298C、N298V、N298L、N298A、G30 0S、G300A、G300P、G300L、G300W、S303P、S303V、S303C、S303A、S303R、N304T、 N304R, N304Q, N304L, N304V, T309G, T309I, T309R, T309M, E314Y, E314T, E314V, E314G, E314S, E314L, E314A, Q318L, Q318R, T326V, T326G, T326W, T326 N、T326A、V330S、V330L、V330P、V330R、V330A、V330G、E342M、E342W、E342N、 E342L、E342R、S343R、S343C、E348W、E348F、E348P、E348V、E348G、E348M、T35 2P, T352L, T352G, T352Q, T352Y, Q359K, Q359P, Q359R, Q359S, Q359A, S371A, S371G, T378G, T378L, T378D, T378H, T378A, T378P, I383A, I383G, I383C, I 383L, I383T, I383M, F386S, F386W, F386Q, F386V, F386I, F386G, F386C, F386A, F386T, F386L, N394D, N394R, N394Y, N394W, N394E, K396I, K396W, K396PK396Y, K396F, Y408V, Y408E, Y408P, Y408S, Y408K, Y408L, K410S, K410R, D412M, D412S, D412N, D412W, D412L, D412R, S417Y, V41 9S, V419G, V419C, V419A, V419K, V419R, V419T, A426M, A426N, A426K, A426R, S427G, S427A, S427P, S427N, S427D, S427L, A434Q, A434G, Q439W, Q439S, Q439G, Q439C, Q439R, Q439Y, G442V, G442D, G442C, G442A, G442L, G442W, G442E, G442M, G442R, L448G, L44 8P, L448E, N470H, N470D, N470K, N470V, N470L, E472I, W475P, W475A, W475R, V485A, V485W, V485K, V485G, V485R, D486I, D486K, D486Y, D486S, D486A, D486W, D486L, S492L, S492R, S492T, S492W, S492P, S492C, A493V, A493R, A493D, A493W, D494N, D494R, D49 4G, D494L, D494E, D494Q, N495L, N495W, N495G, N495R, N495C, I509E, I509D, I509S, I509F, I509W, I509R, T510F, T510E, T510R, T510P, T510V, T510A, T510L, I519W, I519L, I519R, I519F, I519K, N527T, N527K, N527P, N527L, P537D, P537M, P537W, P537G, P537E, N538D, N538S, N538W, N538Y, N538A, S546P, S546G, S546C, S546E, S546N, G547D, G547S, G547V, T554Q, and T554G, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in melting temperature of at least 0.9 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, and at least 98% identity with the amino acid sequence having SEQ ID NO: 3.Or at least 99%, but less than 100%, sequence identity.

[0073] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: V18M, V18Q, K28C, K28R, V36K, V36G, V36W, V36A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, V59T, V59S, V59E, S73H, S73R, S73N, S73V, S73G, T86R, T86V, S95A, S95T, S95V, T139D. T139P, T139V, N142Y, N142H, N142C, L145C, L145D, L145G, L145V, L145S, S1 46W, S146L, S146R, S146G, S146P, N147Q, N147V, N147L, N147K, L147D, L147Y , L147H, L147S, N149H, N149T, N149R, N149K, N149S, Y152S, Y152A, Y152R, Y 152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C, Y152W, V153E, V153S, V153 G, V153W, V153Y, T154N, T154R, T154K, T154P, T154V, L157P, L157R, L157A, L157G, L157W, W158T, W158A, W158M, W158V, W158R, W158P, P159S, P159G, P15 9L, P159V, P159A, P159R, P159Q, P159E, S170A, S170P, S170R, S170M, S175L , S175C, S175W, T176R, T176L, T176N, T176A, T176S, T176I, D184P, D184W, D1 84S, D184Y, D184G, S186A, S186R, S186W, R199F, R199E, R199L, R199C, R199 K, A203M, A203W, A203P, A203L, Q210C, Q210G, Q210S, Q210R, Q210L, Q210P, Q 210V, T211R, T211A, T211H, T211K, T211Q, T211G, T211W, T211E, T211I, T21 1V, T211P, T211L, T211D, Q213W, Q213V, Q213D, Q213A, Q213T, Q213R, Q213G,Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W、S248K、S248T、A252E、A252T、A252Y、A252V、A252L、T254D、T254W、T254V、T254G、T254A、L255R、L255Q、L255P、L255G、Y262C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R、S296F、S296L、S296W、S296K、I297S、I297P、I297K、I297F、I297R、I297W、N298W、N298G、N298C、N298V、 N298L、N298A、G300S、G300A、G300P、G300L、G300W、S303P、S303V、S303C、 S303A、S303R、N304T、N304R、N304Q、N304L、N304V、T309G、T309I、T309R、 T309M、E314Y、E314T、E314V、E314G、E314S、E314L、E314A、T326V、T326G、T 326W、T326N、T326A、E342M、E342W、E342N、E342L、E342R、S343R、S343C、T 352P、T352L、T352G、T352Q、T352Y、Q359K、Q359P、Q359R、Q359S、Q359A、I3 83A, I383G, I383C, I383L, I383T, I383M, K396I, K396W, K396P, K396Y, K396F, K410S, K410R, D412M, D412S, D412N, D412W, D412L, D412R, S417Y, V41 9S、V419G、V419C、V419A、V419K、V419R、V419T、A426M、A426N、A426K、A42 6R、S427G、S427A、S427P、S427N、S427D、S427L、A434Q、A434G、Q439W、Q43 9S、Q439G、Q439C、Q439R、Q439Y、N470H、N470D、N470K、N470V、N470L、W47 5P、W475A、W475R、D486I、D486K、D486Y、D486S、D486A、D486W、D486L、S492 L, S492R, S492T, S492W, S492P, S492C, A493V, A493R, A493D, A493W, D494N, D494R, D494G, D494L, D494E, D494Q, N495L, N495W, N495G, N495R, N495C 、T510F、T510E、T510R、T510P、T510V、T510A、T510L、P537D、P537M、P537W 、P537G、P537E、N538D、N538S、N538W、N538Y、N538A、G547D、G547S、G547V、T554Q and T554G, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in melting temperature of at least 1.1 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0074] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: K28C, K28R, T43K, S57P, S57L, S57G, S57F, S57R, S57T, S57A, S73H, S73R, S73N, S73V, S73G, S95A, S95T, S95V, T139D, T139P, T139V, L145C, L145D, L145G, L145V, L145S, N149H, N149T, N149R, N149K, N149S, Y152S, Y152A, Y152R, Y152L, Y152K , Y152E, Y152P, Y152V, Y152I, Y152C, Y152W, V153E, V153S, V153G, V153W, V 153Y, L157P, L157R, L157A, L157G, L157W, W158T, W158A, W158M, W158V, W158 R, W158P, P159S, P159G, P159L, P159V, P159A, P159R, P159Q, P159E, S175L, S175C, S175W, D184P, D184W, D184S, D184Y, D184G, S186A, S186R, S186W, R19 9F, R199E, R199L, R199C, R199K, A203M, A203W, A203P, A203L, Q210C, Q210G , Q210S, Q210R, Q210L, Q210P, Q210V, T211R, T211A, T211H, T211K, T211Q, T2 11G, T211W, T211E, T211I, T211V, T211P, T211L, T211D, Q213W, Q213V, Q213 D. Q213A, Q213T, Q213R, Q213G, Q213S, V214G, V214R, V214W, V214A, V214I, Y 217G, Y217C, Y217A, Y217S, Y217T, Y217F, T218H, T218C, T218A, T218M, T21 8Q, T218G, T219R, T219D, T219S, T219G, T219C, A221V, A221T, A221L, A221P, A221R, A221E, D222V, D222W, D222T, D222G, D222L, D222R, D222N, D222F, D22 2M, L224G, L224D, L224K, L224V, L224R, Y231S, Y231T, Y231R, Y231L, Y231A,Y231V, Y231N, P234D, P234L, P234S, P234V, Y238R, Y238A, Y238Q, Y238C, Y238E, T240C, T240I, T240L, T240S, G244R, G244C, G244P, G244D , G244W, G246L, G246E, G246S, G246R, G246K, G246W, G246D, S248Y, S248P, S248V, S248L, S248F, S248A, S248E, S248W, S248K, S248T, A252 E. A252T, A252Y, A252V, A252L, T254D, T254W, T254V, T254G, T254A, L255R, L255Q, L255P, L255G, Y262C, Y262Q, Y262S, Y262G, Y262V, Y26 2A, Y262W, A271W, A271Y, A271L, K279R, K279W, K279E, K279P, K279G, K279F, L284N, L284Q, L284T, L284S, L284R, L284G, L284V, V294G, V29 4W, V294E, V294S, Y295V, Y295R, S296F, S296L, S296W, S296K, I297S, I297P, I297K, I297F, I297R, I297W, N298W, N298G, N298C, N298V, N2 98L, N298A, N304T, N304R, N304Q, N304L, N304V, S343R, S343C, T352P, T352L, T352G, T352Q, T352Y, K410S, K410R, D412M, D412S, D412N, D 412W, D412L, D412R, V419S, V419G, V419C, V419A, V419K, V419R, V419T, S427G, S427A, S427P, S427N, S427D, S427L, N470H, N470D, N470K, N470V, N470L, W475P, W475A, W475R, S492L, S492R, S492T, S492W, S492P, S492C, A493V, A493R, A493D, A493W, and P537D, P537M, P537W, P537G.P537E, wherein the position corresponds to an amino acid position in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitution at one or more positions provides a glucosylamylase variant having an increase in melting temperature of at least 1.5 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0075] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising a substitution T43K, wherein the position corresponds to an amino acid position in the amino acid sequence shown in SEQ ID NO: 3, and wherein the substitution provides a glucosylamylase variant having an increase in melting temperature of at least 2.0 degrees Celsius, specifically at least 3.0 degrees Celsius, as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence of SEQ ID NO: 3.

[0076] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, G11, I13, K15, A16, V18, L19, N25, S27, S30, A32, A34, V36, V37, S44, S57, V59, F60, Y67, T68, I71, D72, S73, T74, S75, S76, L77, R78, D82, D83, F84, V85, T86, N90, L91, Q93, S95, L101, T102, T 103, S134, L137, T139, N142, L145, S146, N147, Y152, V153, T154, S155, L157, W158, P159, I160, Q162, N163, S170, S175, T176, Y177, S186, R199, A202, A203, T206, Q210, T211, S212, Q213, S215, Q220, A221, D222, N223, L224, F227, P234, S235, Y238, T240, T243, G244, G245, G246, S248, A252, T254, L255, A270, A271, K279, S282, L284, Y295, S296, I297, N298, S299, G300, A302, S303, N304, S316, G319, T326, V330, N339 , E342, S343, Q344, E348, S351, Q359, S362, G363, T365, A366, S371, S372, T378, S381, I383, F386, A392, N394, K396, N401, K410, D412, S414 S417, V419, D420, E433, N437, T438, Q439, F440, G442, A446, N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N495, S501, A502, I509, T510, N512, S516, A518, I519, N527, A530, E534, P537, N538, N539, I541, A545, S546, G547, N552, and T554, wherein the positions correspond to SEQ The amino acid positions in the amino acid sequence shown in ID NO: 3; and the substitutions at one or more of the positions provided by the substitution provide a glucosylamylase variant having at least 1 as measured as an improvement factor IF.The specific activity of 1 is increased, and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0077] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: Q1R, Q1L, Q1T, Q1G, Q1P, Q1K, Q1M, Q1F, Q1S, Q1A, Q1W, S2V, S2Q, S2E, S2D, S2P, S2A, S2T, S2L, S2R, S2K, S2W, S2G, V3G, V3L, V3I, V3A, V3E, D4R, D4C, D4S, D4G, D4N, D4V, D4W, D4F, D4A, S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S8A, S8W, S8 R, S8L, S8Y, S8G, S8M, S8H, S8P, S8Q, S8V, S8C, S8E, S8K, S8T, S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, G11D, I13L, I13A, I13Q, I13S, I13D, I 13R, I13M, I13V, I13G, I13Y, I13E, K15V, K15R, K15I, K15M, K15A, K15F, K15 L, K15S, K15E, K15W, K15G, K15D, A16L, A16V, A16G, A16E, A16S, A16T, A16K, A 16G, V18A, V18R, V18M, V18T, V18L, V18Q, V18I, L19S, L19A, L19K, L19V, L19 C, L19H, L19W, L19F, L19R, N25W, N25Y, N25D, N25F, N25G, N25R, N25V, N25L, N25A, N25S, N25E, N25C, N25Q, S27A, S27W, S27H, S27V, S27T, S27C, S27G, S2 7E, S27L, S27F, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S30T, S30V, A32D, A32E, A32S, A32V, A32R, A32G, A32M, A32T, A32C, A32K, A32W, A34W, A3 4R, A34L, A34Q, A34G, A34C, A34F, A34V, A34E, A34T, A34I, A34P, V36I, V36R, V36A, V36G, V36L, V37C, V37G, V37R, V37A, V37M, S44R, S44W, S44L, S44T, S4 4C, S44A, S44V, S44P, S44E, S57G, S57T, S57H, S57P, S57A, V59T, V59G, V59E,V59Q、V59L、V59R、V59A、F60L、F60S、F60V、F60A、F60I、Y67C、Y67N、Y67A、Y67G、Y67T、Y67V、Y67D、Y67H、Y67R、Y67F、Y67L、Y67P、Y67S、Y67M、T68K、T68C、T68A、T68P、T68R、T68Q、I71T、I71M、I71V、I71S、I71N、I71F、I71D、I71P、I71R、I71L、I71K、D72V、D72L、D72G、D72N、D72R、D72K、D72E、D72W、D72A、D72C、D72Y、D72S、D72Q、D72T、S73A、S73H、S73G、S73N、S73C、S73R、S73V、S73L、S73I、S73W、S73P、T74S、T74E、T74P、T74N、T74F、T74P、T74M、T74R、T74C、S75G、S75N、S75P、S75E、S75C、S75R、S75L、S75K、S75I、S75T、S76H、S76P、S76Q、S76E、L77S、L77Y、L77E、L77A、L77P、R78W、R78G、R78K、R78Q、R78T、R78A、R78C、R78M、R78E、D82V、D82G、D82R、D82N、D82E、D82C、D83L、D83C、D83W、D83A、D83R、D83G、D83V、D83S、D83E、F84Y、F84L、F84S、F84T、F84P、F84E、F84V、F84A、F84W、F84K、F84M、F84R、V85G、V85W、V85P、V85Q、V85E、V85H、V85R、V85T、T86C、T86R、T86G、T86W、T86D、T86V、T86S、T86A、N90G、N90E、N90T、N90P、N90C、L91H、L91P、L91F、L91V、L91R、Q93L、Q93M、Q93C、Q93H、Q93G、Q93R、Q93W、Q93D、Q93A、Q93N、Q93K、S95V、S95R、S95D、S95Y、S95P、S95G、S95Q、S95A、S95K、L101M、L101V、L101R、L101P、L101F、L101H、L101A、L101G、L101N、L101K、L101C、T102N、T102S、T102C、T102R、T102A、T102I、T102M、T102W、T102E、T102P、T102F、T103A、T103S、T103G、T103D、T103I、T103E、T103V、T103N、S134V、S134I、S134M、S134P、S134L、S134A、S134C、L137S、L137D、L137W、L137G、L137R、L137A、L137I、L137T、T139A、T139N、T139S、T139G、T139D、T139H、T139R、N142K、N142E、N142Q、N142R、N142G、N142H、N142W、N142A、L145S、L145W、L145N、L145C、L145V、L145R、L145D、S146V、S146G、S146L、S146T、S146A、S146C、S146P、S146F、S146R、S146W、N147K、N147E、N147S、N147F、N147T、N147I、N147D、N147P、N147Y、N147H、N147L、Y152V、Y152E、Y152L、Y152I、Y152A、Y152M、Y152R、Y152F、Y152G、V153R、V153Y、V153C、T154R、T154G、T154L、T154S、T154A、T154M、T154P、S155R、S155G、S155L、S155A、S155H、S155W、S155C、S155I、S155P、S155M、S155N、S155T、L157P、L157Q、L157V、L157M、L157R、W158R、W158E、W158C、W158K、W158L、W158G、P159S、P159R、P159V、P159Q、P159T、P159D、P159A、P159L、P159G、I160T、I160A、I160V、I160D、I160G、I160S、I160L、I160Y、I160N、I160F、Q162L、Q162K、Q162R、Q162S、Q162H、Q162P、Q162I、Q162V、N163D、N163G、N163R、N163T、N163I、N163Q、N163Y、N163K、N163H、N163W、N163A、N163S、S170A、S175W、S175R、S175T、S175C、T176S、T176R、T176L、T176A、T176W、T176I、Y177S、Y177T、Y177D、Y177V、S186V、S186R、S186E、S186L、S186D、S186C、S186A、S186Q、R199K、R199V、R199A、R199M、R199N、R199W、R199T、R199E、A202S、A202T、A202Q、A202L、A202E、A202P、A202V、A202F、A202W、A202G、A203Q、A203K、A203W、A203R、A203V、A203L、A203M、A203T、A203E、A203G、A203S、A203P、T206I、T206S、T206W、T206V、T206A、T206P、T206G、T206R、Q210D、Q210R、Q210G、Q210A、Q210L、Q210H、Q210P、Q210V、Q210I、Q210C、T211P、T211R、T211S、T211D、T211Q、T211H、T211A、T211L、T211G、T211W、S212V、S212K、S212D、S212T、S212H、S212L、S212P、S212E、S212C、S212A、S212M、Q213Y、Q213D、Q213R、Q213N、Q213S、Q213W、Q213K、Q213L、Q213C、Q213P、S215L、S215T、S215Q、S215R、S215V、S215G、S215N、S215C、Q220L、Q220P、Q220K、Q220R、Q220H、Q220E、A221V、A221T、A221E、A221G、A221P、D222E、D222M、D222A、D222G、D222N、D222V、D222H、N223K、N223R、L224V、F227A、F227V、F227L、F227S、F227Y、F227E、F227G、P234A、P234L、P234Q、P234S、S235C、S235R、S235W、S235G、S235K、Y238C、Y238L、Y238E、Y238W、Y238A、Y238S、Y238G、T240L、T240C、T240G、T240W、T240V、T240R、T240S、T240A、T240E、T243S、T243Q、T243M、T243G、T243L、T243V、T243E、T243P、T243R、T243W、G244W、G244D、G244Y、G244A、G244S、G244R、G245M、G245N、G245S、G245T、G245V、G245D、G245I、G246V、G246W、G246M、G246E、G246N、G246Q、G246S、G246D、G2 46R、S248E、S248L、S248C、S248G、S248P、S248F、S248T、A252S、A252T、A252 V, A252P, A252G, T254A, T254S, T254G, T254P, L255V, L255A, L255P, L255I, L255C, A270W, A270T, A270E, A270C, A270M, A270S, A270L, A270G, A270R, A27 0Y、A270V、A271V、A271R、A271P、A271L、A271W、A271G、A271T、K279V、K279W 、K279A、K279L、K279R、K279E、K279Y、K279P、K279G、K279S、S282G、S282T、S 282L, S282V, S282F, S282R, S282A, S282I, S282W, L284V, L284G, L284S, L284M, L284T, Y295K, Y295H, Y295Q, Y295W, Y295M, Y295F, Y295C, Y295E, Y295V S296A, S296T, S296K, S296N, S296Y, S296F, S296Q, S296P, S296L, S296D, I297L, I297V, I297H, I297R, I297W, I297K, I297T, I297F, I297G, I297Q, N298 M、N298D、N298S、N298R、N298K、N298A、N298V、N298E、N298G、N298L、S299L、 S299G、S299V、S299A、S299R、S299Q、S299M、S299I、S299P、S299T、G300A、G30 0N、G300D、G300R、G300L、G300F、G300C、G300P、G300W、G300T、G300S、A302L 、A302R、A302P、A302V、A302K、A302M、A302Y、A302S、A302T、A302G、S303P、S 303K, S303R, S303C, S303A, S303F, S303W, S303L, S303Q, N304V, N304G, N304P, N304W, N304F, N304E, N304T, N304D, N304R, N304S, N304A, N304I, N304MN304K, S316T, S316C, S316A, S316R, S316P, S316H, S316K, S316F, S316G, S316Q, S316N, S316M, S316L, S316V, G319T, G319R, G319W, G319S, G319Q, G319 A、G319D、T326S、T326G、T326A、T326C、T326Y、T326P、T326I、T326E、T326Q、 V330M、V330G、V330I、V330D、V330P、V330L、V330Y、V330S、V330A、N339T、N33 9R、N339S、N339A、N339Q、N339P、E342L、E342K、E342T、E342M、E342R、E342V 、E342H、E342G、E342Q、E342S、E342F、E342A、E342W、S343A、S343W、S343G、S 343P, S343Q, S343T, S343E, S343R, S343L, Q344L, Q344V, Q344T, Q344D, Q344A, Q344H, Q344K, Q344R, Q344P, Q344E, E348C, E348G, E348V, E348M, E348N E348A、E348I、E348D、E348L、E348K、E348R、S351Y、S351G、S351R、S351C、S3 51N、S351L、S351K、S351V、S351F、S351T、S351A、S351P、S351W、Q359A、Q359 V, Q359T, Q359R, Q359G, Q359L, Q359K, Q359S, Q359P, Q359W, S362V, S362P, S362R, S362G, S362H, S362E, S362M, S362D, S362Y, S362C, S362F, S362A, S36 2Q、G363C、G363H、G363D、G363W、G363R、G363Q、G363S、G363A、G363T、G363P 、T365R、T365W、T365G、T365L、T365C、T365Q、T365I、T365V、T365Y、T365S、T 365E, A366R, A366L, A366I, A366Q, A366P, A366T, A366S, A366E, A366G, A366D, A366W, A366H, S371V, S371R, S371A, S371T, S371G, S371C, S371E, S371PS372P, S372E, S372R, S372A, S372Q, S372N, S372G, S372R, S372L, S372V, S372M, S372C, S372W, T378P, T378A, T378K, T378W, T378M, T378Q, T378G, T378 V、T378E、T378S、T378R、T378L、T378C、T378I、T378D、S381E、S381Y、S381D、 S381N、S381R、S381G、S381V、S381A、S381T、S381P、S381W、S381Q、S381C、S38 1I、I383F、I383N、I383G、I383C、I383E、I383L、I383M、I383V、I383A、I383T 、I383R、I383S、F386L、F386Y、F386R、F386S、F386G、F386M、F386C、F386W、F 386A, A392V, A392R, A392T, A392S, A392E, A392L, A392G, A392P, A392F, A392M, A392I, A392Q, N394A, N394S, N394T, N394R, N394H, N394G, N394C, N394E N394W, N394P, N394L, N394V, N394F, N394Q, N394K, K396S, K396P, K396M, K396F, K396Q, K396E, K396D, K396W, K396L, K396A, K396I, K396R, K396G, K396 C、K396V、N401Q、N401V、N401F、N401S、N401T、N401G、N401R、N401C、N401A、 N401D、N401K、N401E、N401Y、N401W、N401P、N401L、K410S、K410T、K410L、K41 0D、K410M、K410V、K410P、K410N、K410C、K410G、K410Q、K410E、K410W、K410R 、K410H、D412R、D412Q、D412S、D412P、D412E、D412N、D412G、D412V、D412L、D 412W、D412A、D412K、D412M、D412T、S414P、S414A、S414W、S414G、S414L、S41 4R、S414E、S414N、S414T、S414Q、S417R、S417G、S417K、S417Y、S417A、S417N、V419D、V419E、V419A、V419G、V419M、V419L、V419I、D420V、D420A、E433W、E433P、E433M、E433Y、E433S、E433C、E433G、E433A、E433R、E433Q、E433K、N437V、N437E、N437D、N437M、N437T、N437A、N437S、N437W、N437L、N437P、N437Y、N437G、N437Q、N437K、N437R、T438R、T438A、T438K、T438W、Q439A、Q439R、Q439G、Q439W、Q439P、Q439C、Q439M、Q439Y、Q439D、F440T、F440L、F440W、F440E、F440S、G442V、G442L、G442D、G442A、G442C、G442S、G442F、G442M、G442I、G442Y、G442W、A446L、A446R、A446F、A446G、A446S、A446M、A446Q、A446W、A446V、A446P、A446D、N470W、N470G、N470L、N470S、N470P、N470Y、N470A、N470E、N470D、N470H、N470K、N470T、N470M、E472W、E472S、E472L、E472G、E472R、E472P、E472V、E472T、E472K、V474R、V474F、V474Y、V474I、V474M、V474W、V474E、V474Q、V474L、V474G、V474A、V474K、V474T、V474H、W475P、W475S、W475L、W475C、W475Q、W475G、W475R、W475T、N478V、N478A、N478S、N478T、N478R、N478K、N478G、N478L、N478M、N478I、N478D、N478W、N478E、S484Q、S484T、S484E、S484F、S484A、S484G、S484D、S484L、S484W、S484V、S484R、S484Y、S484P、S484M、V485L、V485T、V485A、V485S、V485R、V485G、V485I、V485E、V485D、V485F、V485K、D486I、D486G、D486R、D486E、D486S、D486A、D486T、D486K、D486F、D486M、D486Q、D486C、D486L、D486Y、D486P、A487M、A487E、A487V、A487S、A487C、A487G、S492L、S492P、S492V、S492R、S492Y、S492M、S492H、S492T、S492K、S492W、A493G、A493S、A493Y、A493V、A493T、A493E、A493Q、A493R、D494A、D494S、D494E、D494Q、D494Y、D494G、D494R、D494T、D494W、D494N、D494H、D494L、D494M、D494V、D494P、N495S、N495L、N495F、N495C、N495W、N495R、N495G、S501P、S501T、S501L、S501G、S501M、S501R、S501K、S501V、S501E、S501A、S501C、A502W、A502V、A502S、A502G、A502D、A502E、A502T、A502M、A502Y、A502H、I509G、I509R、I509W、I509A、I509V、I509L、I509S、I509P、I509T、I509E、I509H、I509N、T510R、T510I、T510A、T510H、T510S、T510Y、T510V、T510L、T510K、T510E、T510P、T510F、T510M、N512S、N512Q、N512L、N512G、N512W、N512I、N512M、N512Y、N512K、N512V、N512H、N512F、N512T、N512R、N512D、S516Y、S516R、S516P、S516T、S516G、S516V、S516N、S516L、S516F、S516M、S516A、S516W、S516C、S516K、A518G、A518P、A518W、A518V、A518R、A518L、A518M、A518F、A518Y、A518S、I519L、I519C、I519G、I519W、I519S、I519Y、I519N、I519A、I519V、I519Q、I519T、I519H、I519M、N527S、N527L、N527V、N527G、N527W、N527H、N527R、N527K、A530R、A530C、A530S、A530G、A530F、A530Y、A530W、A530T、A530V, E534M, E534A, E534V, E534W, E534C, E534R, E534T, E534L, E534G , E534F, E534S, E534Q, E534K, P537R, P537T, P537H, P537M, P537G, P537 A. P537S, P537E, P537Y, P537L, P537V, N538G, N538V, N538R, N538A, N53 8W, N538D, N538M, N538S, N538I, N538Y, N539L, N539S, N539A, N539I, N53 9V, I541A, I541G, I541T, I541W, I541K, I541V, I541N, I541F, A545L, A545W, A545V, A545S, A545G, A545R, A545T, A545P, S546E, S546C, S546G, S546N, S546V, G547S, G547V, G547L, G547D, G547R, G547C, G547M, N552V, N552E, N552D, N552G and T554A, T554G, T554E, T554D, T554C, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more positions thereon provide a glucosylamylase variant having an increase in specific activity of at least 1.1 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0078] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: Q1, V3, D4, S5, S8, S9, I13, A16, V18, S27, S30, A32, V37, S44, S57, V59, F60, Y67, I71, S73, T74, S75, L77, R78, F84, V85, T86, Q93, S95, L101, T102, T103, L137, N142, N147, Y152, V153, L157, W158, N163, S1 86, R199, Q210, T211, S212, S215, N223, L224, P234, S235, T240, T243, T254, K279, S282, I297, N298, S299, G300, S303, E342, S343, Q344, E348, G363, A366, S381, A392, Q439, V474, W475, D486, A493, A502, T510, A518, I519, A530, E534, N538 and S546, G547, wherein the positions correspond to in SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more of the positions provide a glucosylamylase variant having an increase in specific activity of at least 1.2 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0079] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions selected from the group consisting of: Q1R, Q1L, Q1T, Q1G, Q1P, Q1K, Q1M, Q1F, Q1S, Q1A, Q1W, V3G, V3L, V3I, V3A, V3E, D4R, D4C, D4S, D4G, D4N, D4V, D4W, D4F, D4A, S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S8A, S8W, S8R, S8L, S8Y, S8G, S8M, S8H, S8P, S8Q, S8V, S8C, S8E, S8K, S8T , S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, I13L, I13A, I13Q, I1 3S, I13D, I13R, I13M, I13V, I13G, I13Y, I13E, A16L, A16V, A16G, A16E, A16S, A16T, A16K, A16G, V18A, V18R, V18M, V18T, V18L, V18Q, V18I, S27A, S27W, S2 7H, S27V, S27T, S27C, S27G, S27E, S27L, S27F, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S30T, S30V, A32D, A32E, A32S, A32V, A32R, A32G, A32M, A3 2T, A32C, A32K, A32W, V37C, V37G, V37R, V37A, V37M, S44R, S44W, S44L, S44T, S44C, S44A, S44V, S44P, S44E, S57G, S57T, S57H, S57P, S57A, V59T, V59G, V5 9E, V59Q, V59L, V59R, V59A, F60L, F60S, F60V, F60A, F60I, Y67C, Y67N, Y67A, Y67G, Y67T, Y67V, Y67D, Y67H, Y67R, Y67F, Y67L, Y67P, Y67S, Y67M, I71T, I7 1M, I71V, I71S, I71N, I71F, I71D, I71P, I71R, I71L, I71K, S73A, S73H, S73G, S73N, S73C, S73R, S73V, S73L, S73I, S73W, S73P, T74S, T74E, T74P, T74N, T7 4F, T74P, T74M, T74R, T74C, S75G, S75N, S75P, S75E, S75C, S75R, S75L, S75K,S75I、S75T、L77S、L77Y、L77E、L77A、L77P、R78W、R78G、R78K、R78Q、R78T、R78A、R78C、R78M、R78E、F84Y、F84L、F84S、F84T、F84P、F84E、F84V、F84A、F84W、F84K、F84M、F84R、V85G、V85W、V85P、V85Q、V85E、V85H、V85R、V85T、T86C、T86R、T86G、T86W、T86D、T86V、T86S、T86A、Q93L、Q93M、Q93C、Q93H、Q93G、Q93R、Q93W、Q93D、Q93A、Q93N、Q93K、S95V、S95R、S95D、S95Y、S95P、S95G、S95Q、S95A、S95K、L101M、L101V、L101R、L101P、L101F、L101H、L101A、L101G、L101N、L101K、L101C、T102N、T102S、T102C、T102R、T102A、T102I、T102M、T102W、T102E、T102P、T102F、T103A、T103S、T103G、T103D、T103I、T103E、T103V、T103N、L137S、L137D、L137W、L137G、L137R、L137A、L137I、L137T、N142K、N142E、N142Q、N142R、N142G、N142H、N142W、N142A、N147K、N147E、N147S、N147F、N147T、N147I、N147D、N147P、N147Y、N147H、N147L、Y152V、Y152E、Y152L、Y152I、Y152A、Y152M、Y152R、Y152F、Y152G、V153R、V153Y、V153C、L157P、L157Q、L157V、L157M、L157R、W158R、W158E、W158C、W158K、W158L、W158G、N163D、N163G、N163R、N163T、N163I、N163Q、N163Y、N163K、N163H、N163W、N163A、N163S、S186V、S186R、S186E、S186L、S186D、S186C、S186A、S186Q、R199K、R199V、R199A、R199M、R199N、R199W、R199T、R199E、Q210D、Q210R、Q210G、Q210A、Q210L、Q210H、Q210P、Q210V、Q210I、Q210C、T211P、T211R、T211S、T211D、T211Q、T2 11H、T211A、T211L、T211G、T211W、S212V、S212K、S212D、S212T、S212H、S212 L、S212P、S212E、S212C、S212A、S212M、S215L、S215T、S215Q、S215R、S215V、 S215G、S215N、S215C、N223K、N223R、L224V、P234A、P234L、P234Q、P234S、S23 5C、S235R、S235W、S235G、S235K、T240L、T240C、T240G、T240W、T240V、T240R 、T240S、T240A、T240E、T243S、T243Q、T243M、T243G、T243L、T243V、T243E、T 243P, T243R, T243W, T254A, T254S, T254G, T254P, K279V, K279W, K279A, K279L, K279R, K279E, K279Y, K279P, K279G, K279S, S282G, S282T, S282L, S282V S282F, S282R, S282A, S282I, S282W, I297L, I297V, I297H, I297R, I297W, I297K, I297T, I297F, I297G, I297Q, N298M, N298D, N298S, N298R, N298K, N298 A、N298V、N298E、N298G、N298L、S299L、S299G、S299V、S299A、S299R、S299Q、 S299M、S299I、S299P、S299T、G300A、G300N、G300D、G300R、G300L、G300F、G30 0C、G300P、G300W、G300T、G300S、S303P、S303K、S303R、S303C、S303A、S303F 、S303W、S303L、S303Q、E342L、E342K、E342T、E342M、E342R、E342V、E342H、E 342G, E342Q, E342S, E342F, E342A, E342W, S343A, S343W, S343G, S343P, S343Q, S343T, S343E, S343R, S343L, Q344L, Q344V, Q344T, Q344D, Q344A, Q344HQ344K, Q344R, Q344P, Q344E, E348C, E348G, E348V, E348M, E348N, E348A, E348I, E348D, E348L, E348K, E348R, G363C, G363H, G363D, G363W, G363R, G363 Q, G363S, G363A, G363T, G363P, A366R, A366L, A366I, A366Q, A366P, A366T, A366S, A366E, A366G, A366D, A366W, A366H, S381E, S381Y, S381D, S381N, S38 1R, S381G, S381V, S381A, S381T, S381P, S381W, S381Q, S381C, S381I, A392V, A392R, A392T, A392S, A392E, A392L, A392G, A392P, A392F, A392M, A392I, A 392Q、Q439A、Q439R、Q439G、Q439W、Q439P、Q439C、Q439M、Q439Y、Q439D、V47 4R、V474F、V474Y、V474I、V474M、V474W、V474E、V474Q、V474L、V474G、V474A、 V474K、V474T、V474H、W475P、W475S、W475L、W475C、W475Q、W475G、W475R、W4 75T、D486I、D486G、D486R、D486E、D486S、D486A、D486T、D486K、D486F、D486 M、D486Q、D486C、D486L、D486Y、D486P、A487M、A493G、A493S、A493Y、A493V、 A493T、A493E、A493Q、A493R、A502W、A502V、A502S、A502G、A502D、A502E、A50 2T、A502M、A502Y、A502H、T510R、T510I、T510A、T510H、T510S、T510Y、T510V 、T510L、T510K、T510E、T510P、T510F、T510M、A518G、A518P、A518W、A518V、A 518R、A518L、A518M、A518F、A518Y、A518S、I519L、I519C、I519G、I519W、I51 9S、I519Y、I519N、I519A、I519V、I519Q、I519T、I519H、I519M、A530R、A530C、A530S, A530G, A530F, A530Y, A530W, A530T, A530V, E534M, E534A, E534V, E534W, E534C, E534R, E534T, E534L, E534G, E534F, E534S, E534Q, E534K, N538G, N538V, N538R, N538A, N538W, N538D, N538M, N538S, N538I, N538Y, S546E, S546C, S546G, S546N and S546V, G547S, G547V, G547L, G547D, G547R, G547C, G547M, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitution at one or more positions provides a glucosylamylase variant having an increase in specific activity of at least 1.2 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0080] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions selected from the group consisting of: Q1, S5, S9, I13, V18, S30, V37, V59, I71, S73, T74, F84, V85, Q93, L137, N142, W158, N163, S186, S215, N223, P234, S235, S299, E348, Q439, D486, A493, E534, and S546, wherein the majority of positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in specific activity of at least 1.3 as measured by an improvement factor IF, and further wherein the variant is associated with a SEQ ID NO: The amino acid sequence of 3 has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0081] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: Q1R, Q1L, Q1T, Q1G, Q1P, Q1K, Q1M, Q1F, Q1S, Q1A, Q1W, S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, I13L, I13A, I13Q, I13S, I13D, I13R, I13M, I13V, I13G, I13Y, I13E, V18A, V18R, V18M, V 18T, V18L, V18Q, V18I, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S30 T, S30V, V37C, V37G, V37R, V37A, V37M, V59T, V59G, V59E, V59Q, V59L, V59R, V 59A, I71T, I71M, I71V, I71S, I71N, I71F, I71D, I71P, I71R, I71L, I71K, S73 A. S73H, S73G, S73N, S73C, S73R, S73V, S73L, S73I, S73W, S73P, T74S, T74E, T 74P, T74N, T74F, T74P, T74M, T74R, T74C, F84Y, F84L, F84S, F84T, F84P, F84 E, F84V, F84A, F84W, F84K, F84M, F84R, V85G, V85W, V85P, V85Q, V85E, V85H, V 85R, V85T, Q93L, Q93M, Q93C, Q93H, Q93G, Q93R, Q93W, Q93D, Q93A, Q93N, Q93 K, L137S, L137D, L137W, L137G, L137R, L137A, L137I, L137T, N142K, N142E, N 142Q, N142R, N142G, N142H, N142W, N142A, W158R, W158E, W158C, W158K, W15 8L, W158G, N163D, N163G, N163R, N163T, N163I, N163Q, N163Y, N163K, N163H, N163W, N163A, N163S, S186V, S186R, S186E, S186L, S186D, S186C, S186A, S18 6Q, S215L, S215T, S215Q, S215R, S215V, S215G, S215N, S215C, N223K, N223R,P234A, P234L, P234Q, P234S, S235C, S235R, S235W, S235G, S235K, S299L, S 299G, S299V, S299A, S299R, S299Q, S299M, S299I, S299P, S299T, E348C, E34 8G, E348V, E348M, E348N, E348A, E348I, E348D, E348L, E348K, E348R, Q439 A. Q439R, Q439G, Q439W, Q439P, Q439C, Q439M, Q439Y, Q439D, D486I, D486G, D486R, D486E, D486S, D486A, D486T, D486K, D486F, D486M, D486Q, D486C, D486L, D486Y, D486P, A493G, A493S, A493Y, A493V, A493T, A493E, A493Q, A493R, E534M, E534A, E534V, E534W, E534C, E534R, E534T, E534L, E534G, E534F, E534S, E534Q, E534K, S546E, S546C, S546G, S546N, and S546V, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in specific activity of at least 1.3 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0082] In another embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions selected from the group consisting of: S5, S9, S30, V37, S73, F84, V85, S186, S215, Q439, D486, E534, and S546, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in specific activity of at least 1.5 as measured by an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95% sequence identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, of the amino acid sequence having SEQ ID NO: 3.

[0083] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S30T, S 30V, V37C, V37G, V37R, V37A, V37M, S73A, S73H, S73G, S73N, S73C, S73R, S73V, S73L, S73I, S73W, S73P, F84Y, F8 4L, F84S, F84T, F84P, F84E, F84V, F84A, F84W, F84K, F84M, F84R, V85G, V85W, V85P, V85Q, V85E, V85H, V85R, V85 T, S186V, S186R, S186E, S186L, S186D, S186C, S186A, S186Q, S215L, S215T, S215Q, S215R, S215V, S215G, S215 N, S215C, Q439A, Q439R, Q439G, Q439W, Q439P, Q439C, Q439M, Q439Y, Q439D, D486I, D486G, D486R, D486E, D486S D486A, D486T, D486K, D486F, D486M, D486Q, D486C, D486L, D486Y, D486P, E534M, E534A, E534V, E534W, E534C, E534R, E534T, E534L, E534G, E534F, E534S, E534Q, E534K, S546E, S546C, S546G, S546N, and S546V, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitution at one or more of the positions provides a glucosylamylase variant having an increase in specific activity of at least 1.5 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0084] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: Q1R, Q1L, Q1T, Q1G, Q1P, Q1K, Q1M, Q1F, Q1S, Q1A, Q1W, S2V, S2Q, S2E, S2D, S2P, S2A, S2T, S2L, S2R, S2K, S2W, S2G, V3G, V3L, V3I, V3A, V3E, D4R, D4C, D4S, D4G, D4N, D4V, D4W, D4F, D4A, S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S8A, S8W, S8 R, S8L, S8Y, S8G, S8M, S8H, S8P, S8Q, S8V, S8C, S8E, S8K, S8T, S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, G11D, I13L, I13A, I13Q, I13S, I13D, I 13R, I13M, I13V, I13G, I13Y, I13E, K15V, K15R, K15I, K15M, K15A, K15F, K15 L, K15S, K15E, K15W, K15G, K15D, A16L, A16V, A16G, A16E, A16S, A16T, A16K, A 16G, V18A, V18R, V18M, V18T, V18L, V18Q, V18I, L19S, L19A, L19K, L19V, L19 C, L19H, L19W, L19F, L19R, N25W, N25Y, N25D, N25F, N25G, N25R, N25V, N25L, N25A, N25S, N25E, N25C, N25Q, S27A, S27W, S27H, S27V, S27T, S27C, S27G, S2 7E, S27L, S27F, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S30T, S30V, A32D, A32E, A32S, A32V, A32R, A32G, A32M, A32T, A32C, A32K, A32W, A34W, A3 4R, A34L, A34Q, A34G, A34C, A34F, A34V, A34E, A34T, A34I, A34P, V36I, V36R, V36A, V36G, V36L, V37C, V37G, V37R, V37A, V37M, S44R, S44W, S44L, S44T, S4 4C, S44A, S44V, S44P, S44E, S57G, S57T, S57H, S57P, S57A, V59T, V59E, V59Q,V59L、V59R、、F60S、F60V、F60A、F60I、Y67C、Y67N、Y67A、Y67G、Y67T、Y67V、Y67D、Y67H、Y67R、Y67F、Y67L、Y67P、Y67S、Y67M、T68K、T68C、T68A、T68P、T68R、T68Q、I71T、I71M、I71V、I71S、I71N、I71F、I71D、I71P、I71R、I71L、I71K、D72L、D72G、D72N、D72R、D72K、D72E、D72W、D72A、D72C、D72Y、D72S、D72Q、D72T、S73H、S73G、S73N、S73C、S73R、S73V、S73L、S73I、S73W、S73P、T74S、T74E、T74P、T74N、T74F、T74P、T74M、T74R、T74C、S75G、S75N、S75P、S75E、S75C、S75R、S75L、S75K、S75I、S75T、S76H、S76P、S76Q、S76E、L77S、L77Y、L77E、L77P、R78W、R78G、R78K、R78Q、R78T、R78A、R78C、R78M、R78E、D82V、D82G、D82R、D82N、D82E、D82C、D83L、D83C、D83W、D83A、D83R、D83G、D83V、D83S、D83E、F84Y、F84L、F84S、F84T、F84P、F84E、F84V、F84A、F84W、F84K、F84M、F84R、V85G、V85W、V85P、V85Q、V85E、V85H、V85R、V85T、T86C、T86R、T86G、T86W、T86D、T86V、T86S、T86A、N90G、N90E、N90T、N90P、N90C、L91H、L91P、L91F、L91V、L91R、Q93L、Q93M、Q93C、Q93H、Q93G、Q93R、Q93W、Q93D、Q93A、Q93N、Q93K、S95V、S95R、S95D、S95Y、S95G、S95Q、S95A、S95K、L101M、L101V、L101R、L101P、L101F、L101H、L101A、L101G、L101N、L101K、L101C、T102N、T102S、T102C、T102R、T102A、T102I、T102M、T102W、T102E、T102P、T102F、T103A、T103S、T103G、T103D、T103I、T103E、T103V、T103N、S134V、S134I、S134M、S134P、S134L、S134A、S134C、L137S、L137D、L137W、L137G、L137R、L137A、L137I、L137T、T139A、T139N、T139S、T139G、T139D、T139H、T139R、N142K、N142E、N142Q、N142R、N142G、N142H、N142W、N142A、L145S、L145W、L145N、L145C、L145V、L145R、L145D、S146V、S146G、S146L、S146T、S146A、S146C、S146P、S146F、S146R、S146W、N147K、N147E、N147S、N147F、N147T、N147I、N147D、N147P、N147Y、N147H、N147L、Y152V、Y152E、Y152L、Y152I、Y152A、Y152M、Y152R、Y152F、Y152G、V153R、V153Y、V153C、T154R、T154G、T154L、T154S、T154A、T154M、T154P、S155R、S155G、S155L、S155A、S155H、S155W、S155C、S155I、S155P、S155M、S155N、S155T、L157P、L157Q、L157V、L157M、L157R、W158R、W158E、W158C、W158K、W158L、W158G、P159S、P159R、P159V、P159Q、P159T、P159D、P159A、P159L、P159G、I160T、I160A、I160V、I160D、I160G、I160S、I160L、I160Y、I160N、I160F、Q162L、Q162K、Q162R、Q162S、Q162H、Q162P、Q162I、Q162V、N163D、N163G、N163R、N163T、N163I、N163Q、N163Y、N163K、N163H、N163W、N163A、N163S、S170A、S175W、S175R、S175T、S175C、T176S、T176R、T176L、T176A、T176W、T176I、Y177S、Y177T、Y177D、Y177V、S186V、S186R、S186E、S186L、S186D、S186C、S186A、S186Q、R199K、R199V、R199A、R199M、R199N、R199W、R199T、R199E、A202S、A202T、A202Q、A202L、A202E、A202P、A202V、A202F、A202W、A202G、A203Q、A203K、A203W、A203R、A203V、A203L、A203M、A203T、A203E、A203G、A203S、A203P、T206I、T206S、T206W、T206V、T206A、T206P、T206G、T206R、Q210D、Q210R、Q210G、Q210A、Q210L、Q210H、Q210P、Q210V、Q210I、Q210C、T211P、T211R、T211S、T211D、T211Q、T211H、T211A、T211L、T211G、T211W、S212V、S212K、S212D、S212T、S212H、S212L、S212P、S212E、S212C、S212A、S212M、Q213Y、Q213D、Q213R、Q213N、Q213S、Q213W、Q213K、Q213L、Q213C、Q213P、S215L、S215T、S215Q、S215R、S215V、S215G、S215N、S215C、Q220L、Q220P、Q220K、Q220R、Q220H、Q220E、A221V、A221T、A221E、A221G、A221P、D222E、D222M、D222A、D222G、D222N、D222V、D222H、N223K、N223R、L224V、F227A、F227V、F227L、F227S、F227Y、F227E、F227G、P234A、P234L、P234Q、P234S、S235C、S235R、S235W、S235G、S235K、Y238C、Y238L、Y238E、Y238W、Y238A、Y238S、Y238G、T240L、T240C、T240G、T240W、T240V、T240R、T240S、T240A、T240E、T243S、T243Q、T243M、T243G、T243L、T243V、T243E、T243P、T243R、T243W、G244W、G244D、G244Y、G244A、G244S、G244R、G245M、G245N、G245S、G245T、G245V、G245D、G245I、G246V、G246W、G246M、G246E、G246N、G246Q、G246S、G246D、G246R、S248E、S248L、S248C、S248G、S2 48P、S248F、S248T、A252S、A252T、A252V、A252P、A252G、T254A、T254S、T254 G、T254P、L255V、L255A、L255P、L255I、L255C、A270W、A270T、A270E、A270C、 A270M、A270S、A270L、A270G、A270R、A270Y、A270V、A271R、A271P、A271L、A27 1W、A271G、A271T、K279V、K279W、K279A、K279L、K279R、K279E、K279Y、K279P 、K279G、K279S、S282G、S282T、S282L、S282V、S282F、S282R、S282A、S282I、S 282W, L284V, L284G, L284S, L284M, L284T, Y295K, Y295H, Y295Q, Y295W, Y295M, Y295F, Y295C, Y295E, Y295V, S296A, S296T, S296K, S296N, S296Y, S296F S296Q, S296P, S296L, S296D, I297L, I297V, I297H, I297R, I297W, I297K, I297T, I297F, I297G, I297Q, N298M, N298D, N298S, N298R, N298K, N298A, N298 V、N298E、N298G、N298L、S299L、S299G、S299V、S299A、S299R、S299Q、S299M、 S299I、S299P、S299T、G300A、G300N、G300D、G300R、G300L、G300F、G300C、G30 0P, G300W, G300T, G300S, A302L, A302R, A302P, A302V, A302K, A302M, A302Y, A302S, A302T, A302G, S303P, S303K, S303R, S303C, S303A, S303F, S303W, S 303L, S303Q, N304V, N304G, N304P, N304W, N304F, N304E, N304T, N304D, N304R, N304S, N304A, N304I, N304M, N304K, S316T, S316C, S316A, S316R, S316PS316H, S316K, S316F, S316G, S316Q, S316N, S316M, S316L, S316V, G319T, G319R, G319W, G319S, G319Q, G319A, G319D, T326S, T326G, T326A, T326C, T326 Y、T326P、T326I、T326E、T326Q、V330M、V330G、V330I、V330D、V330P、V330L、 V330Y、V330S、V330A、N339T、N339R、N339S、N339A、N339Q、N339P、E342L、E34 2K, E342T, E342M, E342R, E342V, E342H, E342G, E342Q, E342S, E342F, E342A, E342W, S343A, S343W, S343G, S343P, S343Q, S343T, S343E, S343R, S343L, Q 344L、Q344V、Q344T、Q344D、Q344A、Q344H、Q344K、Q344R、Q344P、Q344E、E34 8C、E348G、E348V、E348M、E348N、E348A、E348I、E348D、E348L、E348K、E348R、 S351Y, S351G, S351R, S351C, S351N, S351L, S351K, S351V, S351F, S351T, S351A, S351P, S351W, Q359A, Q359V, Q359T, Q359R, Q359G, Q359L, Q359K, Q359 S、Q359P、Q359W、S362V、S362P、S362R、S362G、S362H、S362E、S362M、S362D、 S362Y、S362C、S362F、S362A、S362Q、G363C、G363H、G363D、G363W、G363R、G36 3Q, G363S, G363A, G363T, G363P, T365R, T365W, T365G, T365L, T365C, T365Q, T365I, T365V, T365Y, T365S, T365E, A366R, A366L, A366I, A366Q, A366P, A 366T, A366S, A366E, A366G, A366D, A366W, A366H, S371V, S371R, S371A, S371T, S371G, S371C, S371E, S371P, S372P, S372E, S372R, S372A, S372Q, S372NS372G, S372R, S372L, S372V, S372M, S372C, S372W, T378P, T378A, T378K, T378W, T378M, T378Q, T378G, T378V, T378E, T378S, T378R, T378L, T378C, T378 I、T378D、S381E、S381Y、S381D、S381N、S381R、S381G、S381V、S381A、S381T、 S381P、S381W、S381Q、S381C、S381I、I383F、I383N、I383G、I383C、I383E、I38 3L, I383M, I383V, I383A, I383T, I383R, I383S, F386L, F386Y, F386R, F386S, F386G, F386M, F386C, F386W, F386A, A392V, A392R, A392T, A392S, A392E, A 392L, A392G, A392P, A392F, A392M, A392I, A392Q, N394A, N394S, N394T, N394R, N394H, N394G, N394C, N394E, N394W, N394P, N394L, N394V, N394F, N394Q N394K, K396S, K396P, K396M, K396F, K396Q, K396E, K396D, K396W, K396L, K396A, K396I, K396R, K396G, K396C, K396V, N401Q, N401V, N401F, N401S, N401 T、N401G、N401R、N401C、N401A、N401D、N401K、N401E、N401Y、N401W、N401P、 N401L、K410S、K410T、K410L、K410D、K410M、K410V、K410P、K410N、K410C、K41 0G、K410E、K410W、K410R、D412R、D412Q、D412S、D412P、D412E、D412N、D412G 、D412V、D412L、D412W、D412A、D412K、D412M、D412T、S414P、S414A、S414W、S 414G、S414L、S414R、S414E、S414N、S414T、S414Q、S417R、S417G、S417K、S41 7Y、S417A、S417N、V419D、V419E、V419A、V419G、V419M、V419L、V419I、D420V、D420A、E433W、E433P、E433M、E433Y、E433S、E433C、E433G、E433A、E433R、E433Q、E433K、N437V、N437E、N437D、N437M、N437T、N437A、N437S、N437W、N437L、N437P、N437Y、N437G、N437Q、N437K、N437R、T438R、T438A、T438K、T438W、Q439A、Q439R、Q439G、Q439W、Q439P、Q439C、Q439M、Q439Y、Q439D、F440T、F440L、F440W、F440E、F440S、G442V、G442L、G442D、G442A、G442C、G442S、G442F、G442M、G442I、G442Y、G442W、A446L、A446R、A446F、A446G、A446S、A446M、A446Q、A446W、A446V、A446P、A446D、N470W、N470G、N470L、N470S、N470P、N470Y、N470A、N470E、N470D、N470H、N470K、N470T、N470M、E472W、E472S、E472L、E472G、E472R、E472P、E472V、E472T、E472K、V474R、V474F、V474Y、V474I、V474M、V474W、V474E、V474Q、V474L、V474G、V474A、V474K、V474T、V474H、W475P、W475S、W475L、W475C、W475Q、W475G、W475R、W475T、N478V、N478A、N478S、N478T、N478R、N478K、N478G、N478L、N478M、N478I、N478D、N478W、N478E、S484Q、S484T、S484E、S484F、S484A、S484G、S484D、S484L、S484W、S484V、S484R、S484Y、S484P、S484M、V485L、V485T、V485A、V485S、V485R、V485G、V485I、V485E、V485D、V485F、V485K、D486I、D486G、D486R、D486E、D486S、D486A、D486T、D486K、D486F、D486M、D486Q、D486C、D486L、D486Y、D486P、A487M、A487E、A487V、A487S、A487C、A487G、S492L、S492P、S492V、S492R、S492Y、S492M、S492H、S492T、S492K、S492W、A493G、A493S、A493Y、A493V、A493T、A493E、A493Q、A493R、D494A、D494S、D494E、D494Q、D494Y、D494G、D494R、D494T、D494W、D494N、D494H、D494L、D494M、D494V、D494P、N495S、N495L、N495F、N495C、N495W、N495R、N495G、S501P、S501T、S501L、S501G、S501M、S501R、S501K、S501V、S501E、S501A、S501C、A502W、A502V、A502S、A502G、A502D、A502E、A502T、A502M、A502Y、A502H、I509G、I509R、I509W、I509A、I509V、I509L、I509S、I509P、I509T、I509E、I509H、I509N、T510R、T510I、T510A、T510H、T510S、T510Y、T510V、T510L、T510K、T510E、T510P、T510F、T510M、N512S、N512Q、N512L、N512G、N512W、N512I、N512M、N512Y、N512K、N512V、N512H、N512F、N512T、N512R、N512D、S516Y、S516R、S516P、S516T、S516G、S516V、S516N、S516L、S516F、S516M、S516A、S516W、S516C、S516K、A518G、A518P、A518W、A518V、A518R、A518L、A518M、A518F、A518Y、A518S、I519L、I519C、I519G、I519W、I519S、I519Y、I519N、I519A、I519V、I519Q、I519T、I519H、I519M、N527S、N527L、N527V、N527G、N527W、N527H、N527R、N527K、A530R、A530C、A530S、A530G、A530F、A530Y、A530W、A530T、A530V、E534M、E534A、E534V、E534W、E534C、E534R、E534T、E534L, E534G, E534F, E534S, E534Q, E534K, P537R, P537T, P537H, P537M, P537G, P537A, P537S, P537E, P537Y, P537L, P537V, N538 G, N538V, N538R, N538A, N538W, N538D, N538M, N538S, N538I, N538Y, N539L, N539S, N539A, N539I, N539V, I541A, I541G, I541T, I54 1W, I541K, I541V, I541N, I541F, A545L, A545W, A545V, A545S, A545G, A545R, A545T, A545P, S546E, S546C, S546G, S546N, S546V, G547S, G547V, G547L, G547D, G547R, G547C, G547M, N552V, N552E, N552D, N552G and T554A, T554G, T554E, T554D, T554C, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more positions thereon provide a glucosylamylase variant having an increase in specific activity of at least 1.1 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0085] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions selected from the group consisting of: Q1R, Q1L, Q1T, Q1G, Q1P, Q1K, Q1M, Q1F, Q1S, Q1A, Q1W, V3G, V3L, V3I, V3A, V3E, D4R, D4C, D4S, D4G, D4N, D4V, D4W, D4F, D4A, S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S8A, S8W, S8R, S8L, S8Y, S8G, S8M, S8H, S8P, S8Q, S8V, S8C, S8E, S8K, S8T , S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, I13L, I13A, I13Q, I1 3S, I13D, I13R, I13M, I13V, I13G, I13Y, I13E, A16L, A16V, A16G, A16E, A16S, A16T, A16K, A16G, V18A, V18R, V18M, V18T, V18L, V18Q, V18I, S27A, S27W, S2 7H, S27V, S27T, S27C, S27G, S27E, S27L, S27F, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S30T, S30V, A32D, A32E, A32S, A32V, A32R, A32G, A32M, A3 2T, A32C, A32K, A32W, V37C, V37G, V37R, V37A, V37M, S44R, S44W, S44L, S44T, S44C, S44A, S44V, S44P, S44E, S57G, S57T, S57H, S57P, S57A, V59T, V59E, V5 9Q, V59L, V59R, F60S, F60V, F60A, F60I, Y67C, Y67N, Y67A, Y67G, Y67T, Y67V, Y67D, Y67H, Y67R, Y67F, Y67L, Y67P, Y67S, Y67M, I71T, I71M, I71V, I71S, I7 1N, I71F, I71D, I71P, I71R, I71L, I71K, S73H, S73G, S73N, S73C, S73R, S73V, S73L, S73I, S73W, S73P, T74S, T74E, T74P, T74N, T74F, T74P, T74M, T74R, T7 4C, S75G, S75N, S75P, S75E, S75C, S75R, S75L, S75K, S75I, S75T, L77S, L77Y,L77E、L77P、R78W、R78G、R78K、R78Q、R78T、R78A、R78C、R78M、R78E、F84Y、F84L、F84S、F84T、F84P、F84E、F84V、F84A、F84W、F84K、F84M、F84R、V85G、V85W、V85P、V85Q、V85E、V85H、V85R、V85T、T86C、T86R、T86G、T86W、T86D、T86V、T86S、T86A、Q93L、Q93M、Q93C、Q93H、Q93G、Q93R、Q93W、Q93D、Q93A、Q93N、Q93K、S95V、S95R、S95D、S95Y、S95G、S95Q、S95A、S95K、L101M、L101V、L101R、L101P、L101F、L101H、L101A、L101G、L101N、L101K、L101C、T102N、T102S、T102C、T102R、T102A、T102I、T102M、T102W、T102E、T102P、T102F、T103A、T103S、T103G、T103D、T103I、T103E、T103V、T103N、L137S、L137D、L137W、L137G、L137R、L137A、L137I、L137T、N142K、N142E、N142Q、N142R、N142G、N142H、N142W、N142A、N147K、N147E、N147S、N147F、N147T、N147I、N147D、N147P、N147Y、N147H、N147L、Y152V、Y152E、Y152L、Y152I、Y152A、Y152M、Y152R、Y152F、Y152G、V153R、V153Y、V153C、L157P、L157Q、L157V、L157M、L157R、W158R、W158E、W158C、W158K、W158L、W158G、N163D、N163G、N163R、N163T、N163I、N163Q、N163Y、N163K、N163H、N163W、N163A、N163S、S186V、S186R、S186E、S186L、S186D、S186C、S186A、S186Q、R199K、R199V、R199A、R199M、R199N、R199W、R199T、R199E、Q210D、Q210R、Q210G、Q210A、Q210L、Q210H、Q210P、Q210V、Q210I、Q210C、T211P、T211R、T211S、T211D、T211Q、T211H、T211A、T211L、T211G、T211W、S2 12V、S212K、S212D、S212T、S212H、S212L、S212P、S212E、S212C、S212A、S212 M、S215L、S215T、S215Q、S215R、S215V、S215G、S215N、S215C、N223K、N223R、 L224V、P234A、P234L、P234Q、P234S、S235C、S235R、S235W、S235G、S235K、T24 0L、T240C、T240G、T240W、T240V、T240R、T240S、T240A、T240E、T243S、T243Q 、T243M、T243G、T243L、T243V、T243E、T243P、T243R、T243W、T254A、T254S、T 254G, T254P, K279V, K279W, K279A, K279L, K279R, K279E, K279Y, K279P, K279G, K279S, S282G, S282T, S282L, S282V, S282F, S282R, S282A, S282I, S282W I297L, I297V, I297H, I297R, I297W, I297K, I297T, I297F, I297G, I297Q, N298M, N298D, N298S, N298R, N298K, N298A, N298V, N298E, N298G, N298L, S299 L、S299G、S299V、S299A、S299R、S299Q、S299M、S299I、S299P、S299T、G300A、 G300N、G300D、G300R、G300L、G300F、G300C、G300P、G300W、G300T、G300S、S30 3P, S303K, S303R, S303C, S303A, S303F, S303W, S303L, S303Q, E342L, E342K, E342T, E342M, E342R, E342V, E342H, E342G, E342Q, E342S, E342F, E342A, E 342W, S343A, S343W, S343G, S343P, S343Q, S343T, S343E, S343R, S343L, Q344L, Q344V, Q344T, Q344D, Q344A, Q344H, Q344K, Q344R, Q344P, Q344E, E348CE348G, E348V, E348M, E348N, E348A, E348I, E348D, E348L, E348K, E348R, G363C, G363H, G363D, G363W, G363R, G363Q, G363S, G363A, G363T, G363P, A366 R, A366L, A366I, A366Q, A366P, A366T, A366S, A366E, A366G, A366D, A366W, A366H, S381E, S381Y, S381D, S381N, S381R, S381G, S381V, S381A, S381T, S38 1P, S381W, S381Q, S381C, S381I, A392V, A392R, A392T, A392S, A392E, A392L, A392G, A392P, A392F, A392M, A392I, A392Q, Q439A, Q439R, Q439G, Q439W, Q 439P、Q439C、Q439M、Q439Y、Q439D、V474R、V474F、V474Y、V474I、V474M、V47 4W、V474E、V474Q、V474L、V474G、V474A、V474K、V474T、V474H、W475P、W475S、 W475L、W475C、W475Q、W475G、W475R、W475T、D486I、D486G、D486R、D486E、D4 86S、D486A、D486T、D486K、D486F、D486M、D486Q、D486C、D486L、D486Y、D486 P、A487M、A493G、A493S、A493Y、A493V、A493T、A493E、A493Q、A493R、A502W、 A502V、A502S、A502G、A502D、A502E、A502T、A502M、A502Y、A502H、T510R、T51 0I、T510A、T510H、T510S、T510Y、T510V、T510L、T510K、T510E、T510P、T510F 、T510M、A518G、A518P、A518W、A518V、A518R、A518L、A518M、A518F、A518Y、A 518S, I519L, I519C, I519G, I519W, I519S, I519Y, I519N, I519A, I519V, I519Q, I519T, I519H, I519M, A530R, A530C, A530S, A530G, A530F, A530Y, A530WA530T, A530V, E534M, E534A, E534V, E534W, E534C, E534R, E534T, E534L, E534G, E534F, E534S, E534Q, E534K, N538G, N538V, N538R, N538A, N538W, N538D, N538M, N538S, N538I, N538Y, S546E, S546C, S546G, S546N and S546V, G547S, G547V, G547L, G547D, G547R, G547C, G547M, wherein the position corresponds to SEQ ID NO: The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more of the positions provide a glucosylamylase variant having an increase in specific activity of at least 1.2 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0086] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more sites selected from the group consisting of: Q1R, Q1L, Q1T, Q1G, Q1P, Q1K, Q1M, Q1F, Q1S, Q1A, Q1W, S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, I13L, I13A, I13Q, I13S, I13D, I13R, I13M, I13V, I13G, I13Y, I13E, V18A, V18R, V18M. V18T, V18L, V18Q, V18I, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S3 0T, S30V, V37C, V37G, V37R, V37A, V37M, V59T, V59E, V59Q, V59L, V59R, I71T, I71M, I71V, I71S, I71N, I71F, I71D, I71P, I71R, I71L, I71K, S73H, S73G, S7 3N, S73C, S73R, S73V, S73L, S73I, S73W, S73P, T74S, T74E, T74P, T74N, T74F, T74P, T74M, T74R, T74C, F84Y, F84L, F84S, F84T, F84P, F84E, F84V, F84A, F8 4W, F84K, F84M, F84R, V85G, V85W, V85P, V85Q, V85E, V85H, V85R, V85T, Q93L , Q93M, Q93C, Q93H, Q93G, Q93R, Q93W, Q93D, Q93A, Q93N, Q93K, L137S, L137D , L137W, L137G, L137R, L137A, L137I, L137T, N142K, N142E, N142Q, N142R, N1 42G, N142H, N142W, N142A, W158R, W158E, W158C, W158K, W158L, W158G, N163 D. N163G, N163R, N163T, N163I, N163Q, N163Y, N163K, N163H, N163W, N163A, N 163S, S186V, S186R, S186E, S186L, S186D, S186C, S186A, S186Q, S215L, S21 5T, S215Q, S215R, S215V, S215G, S215N, S215C, N223K, N223R, P234A, P234L,P234Q, P234S, S235C, S235R, S235W, S235G, S235K, S299L, S299G, S299V, S299A, S299R, S299Q, S299M, S299I, S299P, S299T, E348C, E348G, E348V, E348M, E348N, E348A, E348I, E348D, E348L, E348K, E348R, Q439A, Q439R, Q439G, Q439W, Q439P, Q439C, Q439M, Q439Y, Q439D, D486I, D486G, D486R, D486E, D486S, D486A, D486T, D486K, D486F, D486M, D486Q, D486C, D486L, D486Y, D486P, A493G, A493S, A493Y, A493V, A493T, A493E, A493Q, A493R, E534M, E534A, E534V, E534W, E534C, E534R, E534T, E534L, E534G, E534F, E534S, E534Q, E534K, S546E, S546C, S546G, S546N, and S546V, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in specific activity of at least 1.3 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0087] In one specific embodiment, the present invention relates to a glucosylamylase variant comprising substitutions at one or more positions selected from the group consisting of: 4, 5, 13, 15, 18, 85, specifically substitutions selected from: D4R, S5V, I13S, K15R, V18M, V85G, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3, and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in specific activity as measured as an improvement factor (IF) compared to the glucosylamylase of SEQ ID NO: 3, and further wherein the variant has at least 85%, at least 90%, at least 95% sequence identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, of the amino acid sequence of SEQ ID NO: 3.

[0088] The glucosylamylase variant according to the invention may preferably further contain substitutions corresponding to S95P and A121P, especially S95P + A121P.

[0089] In another embodiment, the glucosylamylase variant of the present invention further comprises a specific combination selected from the following substitutions: S95P + A121P + Y295W, or S95P + A121P + Y295W + Q318Y.

[0090] More specifically, the present invention relates to glucose amylase variants, said variants comprising at least one of the following substitutions or combinations of substitutions: T43K; D4R; S5V; I13S; K15R; V18M; V85G; S95P + A121P + Y295W + T43K; T43K + S95P + A121P + Y295W + Q318Y; V18M + T43K + S95P + A121P + Y295W + Q318Y; D4R + T43K + S95P + A121P + Y295W + Q318Y; S5V + T43K + S95P + A121P + Y295W + Q318Y; I13S + T43K + S95P + A121P + Y295W + Q318Y; V18M + T43K + S95P + A121P + Y295W; The variant is said to have glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 3, and the variant has, compared with the glucosylamylase of SEQ ID NO: 3, an increase in specific activity of at least 1.1 as an improvement factor (IF), and / or an increase in melting temperature of at least 2°C, specifically at least 3°C, as measured by TSA.

[0091] In another specific embodiment, the present invention relates to a glucosylamylase variant comprising at least one of the following substitutions or combinations thereof: T43K; D4R; S5V; I13S; K15R; V18M; V85G; The variant is said to have glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 3, and the variant has an increased specific activity of at least 1.1 compared to the glucosylamylase of SEQ ID NO: 3, and / or an increased melting temperature of at least 2°C, specifically at least 3°C, as measured by TSA.

[0092] In another specific embodiment, the present invention relates to a glucosylamylase variant comprising at least one of the following substitutions or combinations thereof: S95P + A121P + Y295W + T43K; T43K + S95P + A121P + Y295W + Q318Y; V18M + T43K + S95P + A121P + Y295W + Q318Y; D4R + T43K + S95P + A121P + Y295W + Q318Y; S5V + T43K + S95P + A121P + Y295W + Q318Y; I13S + T43K + S95P + A121P + Y295W + Q318Y; V18M + T43K + S95P + A121P + Y295W; The variant is said to have glucosylamylase activity and the variant is said to have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 3, and the variant is said to have a melting temperature that is at least 2°C higher, specifically at least 3°C, as measured by TSA, compared to the glucosylamylase of SEQ ID NO: 3 or the glucosylamylase of SEQ ID NO: 3 having S95P + A121P + Y295W or S95P + A121P + Y295W + Q318Y.

[0093] The variant may further include one or more additional changes at one or more (e.g., several) other locations.

[0094] These amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine fragments, antigenic epitopes, or binding domains.

[0095] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and, for example, by H. Neurath and RL Hill, 1979. The ProteinsThe protein is described in [Protein], Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0096] Alternatively, these amino acid changes have the property of altering the physicochemical properties of the peptide. For example, amino acid changes can improve the peptide's thermal stability, alter substrate specificity, change the optimal pH, etc.

[0097] It can be performed according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science [Science] 244: 1081-1085) to identify essential amino acids in polypeptides. In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the glucosamine activity of the resulting mutant molecule is tested to identify amino acid residues crucial to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. [Journal of Biochemistry] 271: 4699-4708. The active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutations in the amino acids at the putative contact sites. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from comparisons with related peptides.

[0098] In one embodiment, this variant has increased specific activity compared to the parent enzyme. The increased specific activity can be determined as a relative specific activity as described in the acarbose assay in the examples herein.

[0099] In one embodiment, this variant exhibits increased thermal stability compared to the parent enzyme. Thermal stability can be determined by TSA assay, as described in the examples herein.

[0100] Parental glucosidase In one embodiment, the parental glucosyl amylase is derived from the genus *Pleurotus* (…). Gloeophyllum Specifically, it is *Cladosporium hygroscopicum* (…). Gloeophyllum sepiarium ) 。 The parental glucosylamylase may be (a) a polypeptide having at least 85% sequence identity with the polypeptide of SEQ ID NO: 3; (b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the coding sequence of the mature polypeptide of SEQ ID NO: 1, or (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide that has at least 85% sequence identity with the coding sequence of the mature polypeptide of SEQ ID NO: 1.

[0101] In one aspect, the parent has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO: 3, said polypeptide having glucosylamylase activity. In another aspect, the amino acid sequence of the parent differs from that of the polypeptide of SEQ ID NO: 3 by up to 10 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0102] In another respect, the parent contains or consists of the amino acid sequence of SEQ ID NO: 3.

[0103] In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 3.

[0104] In another respect, the parent is encoded by a polynucleotide that hybridizes under high-strict or very high-strict conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, or (ii) or the full-length complement of (i) (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

[0105] The polynucleotide or subsequence of SEQ ID NO: 1 and the polypeptide or fragment thereof of SEQ ID NO: 3 can be used to design nucleic acid probes to identify and clone parental DNA from strains of different genera or species according to methods well known in the art. Specifically, such probes can be used to hybridize with genomic DNA or cDNA of cells of interest, following standard DNA blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be significantly shorter than the complete sequence, but should be at least 15, for example at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, for example at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, or at least 900 nucleotides. Both DNA and RNA probes can be used. Typically, the probes are labeled (e.g., with...). 32 P, 3 H, 35 (S, biotin, or avidin) to detect the corresponding gene. This invention covers such probes.

[0106] DNA that hybridizes to the probes described above and encodes the parent can be screened from genomic DNA or cDNA libraries prepared from other strains of this type. Genomic DNA or other DNA from these other strains can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from the library or separated DNA can be transferred and immobilized on nitrocellulose or other suitable vector materials. This vector material is used in DNA blotting to identify clones or DNA that hybridize to SEQ ID NO: 1 or its subsequences.

[0107] For the purposes of this invention, hybridization refers to the hybridization of a polynucleotide with a nucleic acid probe labeled with the following: (i) SEQ ID NO: 1; (ii) the coding sequence of the mature polypeptide of SEQ ID NO: 1; (iii) its full-length complementary sequence; or (v) its subsequence. Hybridization is performed under very low to very high stringent conditions. Molecules hybridized with nucleic acid probes under these conditions can be detected using, for example, X-ray film or any other detection method known in the art.

[0108] In one aspect, the nucleic acid probe is the coding sequence of the mature polypeptide of SEQ ID NO: 1. In another aspect, the nucleic acid probe is nucleotides 52 to 1719 of SEQ ID NO: 1. In yet another aspect, the nucleic acid probe is a polynucleotide encoding: the polypeptide of SEQ ID NO: 2; its mature polypeptide; or a fragment thereof. In yet another aspect, the nucleic acid probe is SEQ ID NO: 1.

[0109] In another embodiment, the parent is encoded by a polynucleotide having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO: 1.

[0110] The polypeptide can be a hybrid polypeptide, in which a region of one polypeptide is fused to the N-terminus or C-terminus of another polypeptide region.

[0111] The parent peptide can be a fusion peptide or a cleavable fusion peptide, wherein another peptide is fused to the N-terminus or C-terminus of the peptide of the present invention. Fusion peptides are generated by fusing a polynucleotide encoding another peptide with a polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking the coding sequences of the peptides such that they are within a frame and that the expression of the fusion peptide is under the control of one or more identical promoters and terminators. Fusion peptides can also be constructed using integrin technology, in which the fusion peptide is generated post-translational (Cooper et al., 1993, ). EMBO J. [Journal of the European Society for Molecular Biology] 12: 2575-2583; Dawson et al., 1994. Science [Science] 266: 776-779.

[0112] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved during the secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology and Biotechnology] 3: 568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63: 3488-3493; Ward et al., 1995, Biotechnology [Biotechnology] 13: 498-503; and Contreras et al., 1991, Biotechnology [Biotechnology] 9: 378-381; Eaton et al., 1986, Biochemistry [Biochemistry] 25: 505-512; Collins-Racie et al., 1995, Biotechnology [Biotechnology] 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and (Stevens, 2003, Drug Discovery World 4: 35-48).

[0113] The parent can be a fungal glucosylamylase. For example, the parent can be a glucosylamylase from the genus Myxobolus or Myxobolus.

[0114] In another aspect, the parent is *Clostridium perfringens*, *Clostridium hygroscopicum*, or *Clostridium valvulpis* glucosylamylase.

[0115] In another respect, the parent is a glucosyl amylase from *Entoloma hygroscopicum*, such as the glucosyl amylase of SEQ ID NO: 2 or a mature polypeptide as disclosed in SEQ ID NO: 3.

[0116] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Netherlands Culture Collection (CentraalbureauVoor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Patent Culture Collection of the Agricultural Research Service.

[0117] The parent can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from their natural environment are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening another microorganism or a library of genomic DNA or cDNA from a mixed DNA sample. Once the polynucleotide encoding the parent has been detected with the probe, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, see above).

[0118] Preparation of variants These variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0119] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[0120] Site-directed mutagenesis can be achieved in vitro via PCR involving primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent and subsequent ligation of the mutated oligonucleotide into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide, thus allowing the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. [Nucleic Acids Research] 18: 7349-4966.

[0121] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

[0122] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0123] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using various techniques, such as those developed by Tian et al. (2004). Nature The techniques described in [Nature] 432: 1050-1054, based on multi-channel microchips, and similar techniques for synthesizing and assembling oligonucleotides on optically programmable microfluidic chips.

[0124] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures, such as those described by Reidhaar-Olson and Sauer, 1988. Science [Science] 241: 53-57; Bowie and Sauer, 1989. Proc.Natl.Acad.Sci.USA [Proceedings of the National Academy of Sciences] 86: 2152-2156; WO 95 / 17413; or those disclosed in WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry [Biochemistry] 30: 10832-10837; US Patent No. 5,223,409; WO 92 / 06204) and zone-directed mutagenesis (Derbyshire et al., 1986, Gene [Gene] 46: 145; Ner et al., 1988, DNA 7: 127).

[0125] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999). Nature Biotechnology[Nature Biotechnology] 17: 893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0126] Semi-synthetic gene construction is achieved through a combination of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes the process of synthesizing polynucleotide fragments combined with PCR technology. Therefore, defined regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can be amplified using error-prone or non-error-prone PCR. The polynucleotide subsequence can then be shuffled.

[0127] Polynucleotides The present invention also relates to polynucleotides encoding variants of the invention.

[0128] Nucleic acid constructs The present invention also relates to a nucleic acid construct comprising a polynucleotide operably linked to one or more control sequences encoding a variant of the invention, the one or more control sequences guiding the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0129] The polynucleotide can be manipulated in a variety of ways to provide expression of the variant. Depending on the expression vector, manipulation of the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0130] The control sequence can be a promoter, which is a polynucleotide recognized by the host cell for the expression of that polynucleotide. A promoter contains a transcriptional control sequence that mediates the expression of a variant. A promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0131] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells are promoters obtained from the genes of: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucosylamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 96 / 00787). 00 / 56900), Rhizomucormiehei lipase, Rhizomucormiehei aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei Trichoderma β-xylosidase, and the NA2-tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene, wherein the untranslated pre-progenitor is replaced by an untranslated pre-progenitor from the Aspergillus triose phosphate isomerase gene; non-restrictive examples include a modified promoter derived from the Aspergillus niger gene encoding neutral α-amylase, wherein the untranslated pre-progenitor has been replaced by an untranslated pre-progenitor from the Aspergillus nidulans or Aspergillus oryzae gene encoding triose phosphate isomerase); and its mutant, truncated and heterozygous promoters.

[0132] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of a multinucleotide encoding the variant. Any terminator that is functional in the host cell can be used.

[0133] The preferred terminator for filamentous fungal host cells is derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0134] Control sequences can also be mRNA stabilizing regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of that gene.

[0135] This control sequence can also be a leader sequence, a non-translated mRNA region that is important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the polynucleotide encoding that variant. Any leader that functions in the host cell can be used.

[0136] Preferred precursors for use in filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0137] The control sequence can also be a polyadenylation sequence, i.e., a sequence operatively linked to the 3' end of the variant's coding sequence and recognized by the host cell during transcription as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[0138] Preferred polyadenylation of filamentous fungal host cells is obtained from genes targeting the following: Aspergillus nidulans o-aminobenzoate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0139] The control sequence can also be a signal peptide coding region, encoding a signal peptide linked to the N-terminus of the variant and guiding the variant into the cellular secretion pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence, which is naturally linked within the translation reading frame to a segment of the coding sequence of the variant. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is exogenous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the variant. However, any signal peptide coding sequence that guides the expressed variant into the host cell's secretion pathway can be used.

[0140] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus pubescens lipase, and Rhizopus oryzae aspartic protease.

[0141] The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the variant. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. The propeptide-coding sequence can be obtained, for example, from the genes of *Thermophilus thymol* (…). Myceliophthora thermophila laccase (WO 95 / 33836), Rhizopus oryzae ( Rhizomucor miehei Aspartic protease.

[0142] When both the signal peptide sequence and the propeptide sequence are present, the propeptide sequence is located at the N-terminus of the variant immediately adjacent to it, and the signal peptide sequence is located at the N-terminus of the propeptide sequence immediately adjacent to it.

[0143] Also desirable is the addition of regulatory sequences that modulate variant expression relative to host cell growth. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operatively linked to the regulatory sequence.

[0144] expression carrier The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more suitable restriction sites to allow insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector, thereby enabling operative linking of the coding sequence to a suitable control sequence for expression.

[0145] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0146] The vector can be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with the chromosome in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids collectively containing the total DNA of the host cell genome to be introduced, or transposons can be used.

[0147] The vector preferably contains one or more selective markers that allow for easy selection of transformed cells, transfected cells, transduced cells, etc. Selective markers are genes whose products provide biocide or viral resistance, heavy metal resistance, auxotrophic prototrophs, etc.

[0148] The preferred fungi for use in Aspergillus cells are Aspergillus nidus or Aspergillus oryzae. amdS and pyrG Gene.

[0149] The vector preferably contains one or more elements that allow the vector to integrate into the host cell genome or allow the vector to replicate autonomously in the cell independently of the genome.

[0150] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the variant or any other element of the vector used for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides to guide the precise location within the chromosome for integration into the host cell genome via homologous recombination. To increase the likelihood of integration at a precise location, the integrating element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. These integrating elements can be any sequence homologous to the target sequence in the host cell genome. Furthermore, these integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0151] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that functions in the cell to mediate autonomous replication. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0152] Examples of replication origins used in yeast host cells include the 2-micron replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0153] Examples of replication origins used in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987). Nucleic Acids Res [Nucleic Acid Research] 15: 9163-9175; WO 00 / 24883). The AMA1 gene can be isolated and plasmids or vectors containing the gene can be constructed according to the methods disclosed in WO 00 / 24883.

[0154] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase the generation of variants. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene along with the polynucleotide, wherein cells containing an amplified copy of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selectivity reagent.

[0155] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989 (see above)).

[0156] host cells This invention also relates to recombinant host cells containing a polynucleotide operably linked to one or more control sequences encoding variants of the invention, the one or more control sequences directing the generation of variants of the invention. A construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any offspring of a parent cell that is inconsistent with the parent cell due to mutations occurring during replication. The selection of the host cell will depend largely on the gene encoding the variant and its origin.

[0157] The host cell can be any cell that is useful in the recombinant production of variants, such as a eukaryotic cell.

[0158] The host cell can be a eukaryotic organism, such as a fungal cell.

[0159] As used herein, “fungi” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi (as defined by Hawksworth et al., in: Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, International CAB, University Press, Cambridge, UK).

[0160] The fungal host cell can be a yeast cell. As used herein, "yeast" includes *Entomophyceae* (Endosporales), *Basidiomycetes*, and yeasts belonging to the class Deuteromycetes (Bacillus). Because yeast classification may change in the future, for the purposes of this invention, yeast should be defined as described in the biology and activity of yeast (edited by Skinner, Passmore, and Davenport). Soc. App. Bacteriol. Symposium Series As described in Proceedings of the Society for Applied Bacteriology, No. 9 (1980).

[0161] Yeast host cells can be cells from genera such as *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Schizosaccharomyces*, or *Yersinia*, such as *Lactobacillus lactis*. [[ID=49Kluyveromyces lactis ), Kelvin yeast, Saccharomyces cerevisiae, Saccharomyces sacchariformis, Douglas yeast, Klufer yeast, Nodi yeast, Ovoyomyces or Yersinia lipolytica ( [[ID=50Yarrowia lipolytica )cell.

[0162] The host cell for fungi can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms within the subphyla of Eumycota and Oomycota (as described by Hawksworth et al., 1995, see above). Filamentous fungi are typically characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, yeasts (such as Saccharomyces cerevisiae) exhibit vegetative growth through budding of single-celled cells, and carbon metabolism can be fermentative.

[0163] The host cells of filamentous fungi can be *Apertoire*, *Aspergillus*, *Briestrogenus*, or *Cirsium*. Bjerkandera ), *Pseudomonas*, *Aureospora*, *Coprinus*, *Amanita* ( Coriolus Cryptococcus, Ustilagoeaceae ( FilibasidiumFusarium, Pyrophyllus, Mucor, Hymenocytotrichum, Neotrichum, Nematophyte, Penicillium, Penicillium, Leptotrichum, Vespasmidoides Phlebia ), Rumenichthys, Pleurotus ( Pleurotus ), *Schizophyllum*, *Basilaria*, *Thermophila*, *Fusporium*, *Cyclophorus*, *Pteris*, *Pteris* ( Trametes (or Trichoderma cells)

[0164] For example, the host cells of filamentous fungi can be *Aspergillus bubomori*, *Aspergillus stolonifer*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus niger*, *Aspergillus oryzae*, and *Aspergillus niger*. Bjerkandera adusta ), dried cereus ( Ceriporiopsis aneirina ), Carnegie cereus ( Ceriporiopsis caregiea ), Phyllostachys glabra ( Ceriporiopsis gilvescens Panochita cereus ( Ceriporiopsis pannocinta ), Cycloceae ( Ceriporiopsis rivulosa ), *Pseudomonas erythroptera* ( Ceriporiopsis subrufa ), Insectoid cereus ( Ceriporiopsis subvermispora ), Narrow-sided golden spores ( Chrysosporium inops ), keratophilic aureospores, Lukenowens aureospores ( Chrysosporium lucknowense ), Fecal spores ( Chrysosporium merdarium ), *Aureobasidium monnieri*, *Aureobasidium queensii* ( Chrysosporium queenslandicum ), Tropical golden spores, Brown golden spores ( Chrysosporium zonatum ), Grey-covered Ghost Umbrella ( Coprinus cinereus ), scabra ( Coriolus hirsutus Fusarium graminearum, Fusarium graminearum, Fusarium graminearum, Fusarium graminearum, Fusarium graminearum ( Fusarium graminum ), Fusarium heterosporum, Fusarium oxysporum, Fusarium oxysporum, Fusarium multibranchii, Fusarium pinkum, Fusarium elderberry, Fusarium scabiosaefolium, Fusarium fimbriatum, Fusarium sulfideum, Fusarium rotundum, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Mucor spp., Mucor spp., Mucor spp., Mucor spp., Neurospora crassa, Penicillium purpureoides, Pleurotus ostreatus ( Phanerochaete chrysosporium ), Epireobacterium ( Phlebia radiata ), Erycibe and Pleurotus eryngii ( Pleurotus eryngii ), terrestrial closporidium, long-range hairy fungus ( Trametes villosa ), discoloration thrombus ( Trametes versicolor Trichoderma harzianum, Trichoderma cornigliata, Trichoderma longifolia, Trichoderma reesei, or green Trichoderma cells.

[0165] Generation method The present invention also relates to methods for generating variants, the methods comprising: (a) culturing host cells of the present invention under conditions suitable for expressing the variant; and (b) recovering the variant.

[0166] Host cells are cultured in a suitable nutrient medium for variant generation using methods known in the art. For example, cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a laboratory or industrial fermenter under conditions that allow for variant expression and / or isolation. The culture occurs using procedures known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the American Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0167] Variants can be recovered using methods known in the art. For example, variants can be recovered from nutrient media through a variety of routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0168] Variants can be purified to obtain substantially pure variants using a variety of procedures known in the art, including but not limited to chromatographic methods (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification [Protein Purification], edited by Janson and Ryden, VCH Publishers, New York, 1989.

[0169] Alternatively, instead of recycling variants, the host cells of the present invention expressing variants are used as the source of the variants.

[0170] Composition The present invention also relates to compositions comprising the polypeptides of the present invention. Preferably, the composition further comprises a carrier and / or an excipient. More preferably, these compositions are enriched with such a polypeptide. The term "enrichment" indicates that the glucosylamylase activity of the composition has been increased, for example, by an enrichment factor of at least 1.1. Preferably, these compositions are formulated to provide desired properties, such as light color, low odor, and acceptable storage stability.

[0171] The composition may include the glucosylamylase variant of the present invention as the main enzyme component, for example, a single-component composition. Alternatively, the composition may include a variety of enzyme activities, such as aminopeptidase, α-amylase, isoamylase, carbohydrate enzyme, carboxypeptidase, catalase, cellulase, chitosanase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, halogen peroxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinase, peptidase, peroxidase, phytase, polyphenol oxidase, amylopectinase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0172] In one specific embodiment, the composition comprises α-amylase and a variant glucosylase according to the invention. In another embodiment, the composition comprises isoamylase and a variant glucosylase according to the invention. In yet another embodiment, the composition comprises α-amylase, isoamylase, and a variant glucosylase according to the invention.

[0173] In another aspect, the composition comprises a variant glucosylamylase of the present invention in combination with amylopectin. In another aspect, the composition comprises a variant glucosylamylase of the present invention in combination with amylopectin and isoamylase. In yet another aspect, the composition comprises a variant glucosylamylase of the present invention in combination with amylopectin and α-amylase.

[0174] In one specific embodiment, the composition further comprises a protease.

[0175] Peptide compositions can be prepared according to methods known in the art, and these peptide compositions can be in the form of liquid or dry compositions. For example, peptide compositions can be in the form of particles or microparticles. The peptides included in the composition can be stabilized according to methods known in the art.

[0176] Examples of preferred uses of the polypeptides or polypeptide compositions of the present invention are given below. The dosage of the polypeptide compositions of the present invention and other conditions for using the compositions can be determined based on methods known in the art.

[0177] The above compositions are suitable for use in liquefaction, saccharification, and / or fermentation processes, preferably in starch conversion, and especially for the production of syrups and fermentation products, such as ethanol.

[0178] Examples of preferred uses of the polypeptide compositions of the present invention are given below. The dosage of the polypeptide compositions of the present invention and other conditions for using the compositions can be determined based on methods known in the art.

[0179] Method for using the variant glucosylamylase described in this invention - Industrial Application The variant glucosylamylase described in this invention possesses valuable properties that allow for a variety of industrial applications. Specifically, this glucosylamylase can be used in ethanol production and starch conversion processes.

[0180] Variant glucosylamylases can be used in starch processes, particularly starch conversion, especially starch liquefaction (see, for example, U.S. Patent Nos. 3,912,590, EP 252730, EP 063909, WO 99 / 19467, and WO 96 / 28567, all of which are incorporated herein by reference). Compositions for starch conversion purposes, in addition to the glucosylamylase of the present invention, also include α-amylases, pullulanases, and / or proteases.

[0181] Furthermore, the glucosylamylase described in this invention is particularly suitable for producing sweeteners and ethanol (see, for example, U.S. Patent No. 5,231,017, which is incorporated herein by reference), such as fuel ethanol, drinking ethanol, and industrial ethanol, from starch or whole grains.

[0182] In one embodiment, the present invention relates to the use of the glucoamylase according to the invention for producing syrups and / or fermentation products from starch-containing materials. In one embodiment, the starch material may be gelatinized. In another embodiment, the starch material is ungelatinized.

[0183] Starch processing Natural starch consists of microscopic particles that are insoluble in water at room temperature. When an aqueous starch slurry is heated, the particles swell and eventually rupture, dispersing starch molecules into the solution. At temperatures as high as about 50°C to 75°C, the swelling can be reversible. However, at higher temperatures, irreversible swelling begins, a process known as "gelatinization." During this gelatinization process, viscosity increases significantly. The granular starch to be processed can have a highly refined starch quality, preferably at least 90%, at least 95%, at least 97%, or at least 99.5% pure, or it can be a material containing coarser starch, including (e.g., milled) whole grains, which contain non-starch portions such as germ residues and fiber. This raw material (such as whole grains) can be milled, for example, to reduce particle size in order to develop the structure and allow for further processing. In dry milling, the whole grain is milled and used. Wet milling allows for good separation of the germ from the coarse flour (starch granules and protein) and is often used in places where starch hydrolysates are used, for example, in the production of syrups. Both dry and wet milling are well-known starch processing methods in the art and can be used in the method of the present invention. Methods for reducing the particle size of the starch-containing material are known to those skilled in the art.

[0184] Since the solids level in typical industrial processes is 30%-40%, starch must be diluted or "liquefied" to make it suitable for processing. In current commercial practice, this viscosity reduction is mainly achieved through enzymatic degradation.

[0185] Liquefaction is carried out in the presence of α-amylase, preferably bacterial α-amylase and / or acidic fungal α-amylase. In one embodiment, phytase is also present during liquefaction. In another embodiment, viscosity-reducing enzymes such as xylanase and / or β-glucanase are also present during liquefaction.

[0186] During liquefaction, α-amylase degrades long-chain starch into branched and linear shorter units (maltodextrin). Liquefaction can be carried out as a three-step hot slurry method. The slurry is heated to between 60°C and 95°C (e.g., 70°C-90°C, 77°C-86°C, 80°C-85°C, 83°C-85°C) and α-amylase is added to initiate liquefaction (dilution).

[0187] In one embodiment, the slurry can be spray-cooked at 95°C–140°C (e.g., 105°C–125°C) for approximately 1–15 minutes, for example, approximately 3–10 minutes, particularly around 5 minutes. The slurry is then cooled to 60°C–95°C and further α-amylase is added to achieve final hydrolysis (secondary liquefaction). The spray cooking process is typically carried out at a pH of 4.5–6.5, with a pH between 5 and 6. The α-amylase can be added, for example, in a single dose prior to spray cooking.

[0188] The liquefaction process is carried out between 70°C and 95°C, for example, 80°C-90°C, or around 85°C, for approximately 10 minutes to 5 hours, typically 1-2 hours. The pH is between 4 and 7, for example, between 5.5 and 6.2. To ensure optimal enzyme stability under these conditions, calcium may optionally be added (to provide 1-60 ppm of free calcium ions, for example, approximately 40 ppm of free calcium ions). After such treatment, the liquefied starch will typically have a "dextrose equivalent" (DE).

[0189] Generally, liquefaction and liquefaction conditions are well known in the art.

[0190] Examples of α-amylases are disclosed in the “α-amylase” section below.

[0191] Saccharification can be carried out using conditions well known in the art, with enzymes of a carbohydrate-producing source, specifically glucosylamylase or β-amylase, and optionally debranching enzymes (e.g., isoamylase or amylopectinase). For example, the entire saccharification step can last from about 24 to about 72 hours. However, presaccharification, typically 40–90 minutes, is commonly carried out at temperatures between 30°C and 65°C (typically about 60°C), followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF). Saccharification is typically carried out at temperatures in the range of 20°C to 75°C (e.g., 25°C–65°C and 40°C–70°C, typically around 60°C) and at a pH between about 4 and 5, generally at about pH 4.5.

[0192] The saccharification and fermentation steps can be performed sequentially or simultaneously. In one embodiment, saccharification and fermentation are performed simultaneously (referred to as "SSF"). However, typically, a pre-saccharification step is performed at a temperature of 30 to 65°C, typically about 60°C, for about 30 minutes to 2 hours (e.g., 30 to 90 minutes), followed by a complete saccharification during fermentation, known as simultaneous saccharification and fermentation (SSF). The pH is typically between 4.2 and 4.8, for example, pH 4.5. In simultaneous saccharification and fermentation (SSF), there is no maintenance phase of saccharification; in fact, the yeast and enzymes are added together.

[0193] In a typical saccharification process, maltodextrin produced during liquefaction is converted to dextran by adding glucosylamylase and debranching enzymes, such as isoamylase (US Patent No. 4,335,208) or pullulanase. The temperature is lowered to 60°C before adding glucosylamylase and debranching enzymes. The saccharification process takes 24–72 hours. Before adding the saccharifying enzymes, the pH is lowered to below 4.5 while maintaining a high temperature (above 95°C) to inactivate the liquefying α-amylase. This process reduces the formation of short oligosaccharides called "panose precursors," which cannot be properly hydrolyzed by debranching enzymes. Normally, about 0.2%–0.5% of the saccharification product is the branched trisaccharide panose (Glc pα1-6Glc pα1-4Glc), which cannot be degraded by pullulanase. If active amylase from the liquefaction step (i.e., undenatured) is still present during saccharification, the amount of panose can be as high as 1%-2%, which is highly undesirable because it significantly reduces the saccharification yield.

[0194] Other fermentation products may be fermented under conditions and temperatures suitable for the fermenting organisms discussed, as well as those well known to those skilled in the art.

[0195] Fermentation products can be recovered using methods well known in the art (e.g., by distillation).

[0196] In a specific embodiment, the method of the present invention further includes the following steps before converting the starch-containing material into sugar / dextrin: (x) Reduce the particle size of the starch-containing material; and (y) Form a slurry containing the starch-containing material and water.

[0197] In one embodiment, the starch-containing material is milled to reduce particle size. In another embodiment, the particle size is reduced to between 0.05-3.0 mm, preferably 0.1-0.5 mm, or such that at least 30%, preferably at least 50%, more preferably at least 70%, and even more preferably at least 90% of the starch-containing material is suitable for passing through a sieve with a 0.05-3.0 mm screen, preferably a 0.1-0.5 mm screen.

[0198] The aqueous slurry may contain 10 wt.% to 55 wt.% dry solids (DS) of starch-containing material, preferably 25 wt.% to 45 wt.% dry solids (DS), more preferably 30 wt.% to 40 wt.% dry solids (DS).

[0199] Conventional starch conversion processes such as liquefaction and saccharification are described, for example, in U.S. Patent Nos. 3,912,590, EP252730, and EP 063909, which are incorporated herein by reference.

[0200] In an embodiment, the conversion method degrades starch into lower molecular weight carbohydrate components, such as sugar or fat substitutes, and the method includes a debranching step.

[0201] When starch is converted into sugar, it is depolymerized. Such depolymerization processes consist of, for example, a pretreatment step and two or three consecutive process steps, namely, a liquefaction process, a saccharification process, and, depending on the desired end product, an optional isomerization process.

[0202] When the desired final sugar product is, for example, high-fructose corn syrup, dextrose syrup can be converted to fructose. Following the saccharification process, the pH is increased to a value in the range of 6-8, for example, pH 7.5, and calcium is removed by ion exchange. Then, dextrose syrup is converted to high-fructose corn syrup using, for example, an immobilized glucose isomerase.

[0203] Production of fermentation products Fermentable sugars (e.g., dextrins, monosaccharides, especially glucose) are produced by enzymatic saccharification. These fermentable sugars can be further purified and / or converted into useful sugar products. Additionally, these sugars can be used as fermentation feedstocks in microbial fermentation processes to produce end products such as alcohols (e.g., ethanol and butanol), organic acids (e.g., succinic acid, 3-HP, and lactic acid), sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g., lysine), and proteins (e.g., antibodies and their fragments).

[0204] In one embodiment, fermentable sugars obtained during the liquefaction process step are used to produce alcohols, and in particular ethanol. In ethanol production, the SSF process is typically used, in which saccharifying enzymes are added along with fermenting organisms (e.g., yeast), and the process is then carried out at a temperature of 30°C–40°C.

[0205] The organism used in fermentation depends on the desired end product. Typically, yeast is used as the fermentation organism if ethanol is the desired end product. In some preferred embodiments, the ethanol-producing microorganism is yeast, and particularly yeasts such as *Saccharomyces cerevisiae* strains (US Patent No. 4,316,956). Various *Saccharomyces cerevisiae* strains are commercially available and these include (but are not limited to) FALI (Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL (DSM Specialties), REDSTAR (Lesaffre), and Angel Yeast Company (China). The amount of starting yeast used in these methods is an amount that effectively produces a commercially effective amount of ethanol within a suitable time (e.g., producing at least 10% ethanol from a substrate with a DS between 25% and 40% in less than 72 hours). The yeast cells generally ferment at a rate of approximately 10 4 To about 10 12 And preferably from about 10 7 To about 10 10 The yeast is supplied in units of live yeast count per mL of fermentation broth. After adding the yeast to the mash, it is typically allowed to ferment for approximately 24–96 hours, for example, 35–60 hours. The temperature is between approximately 26°C and 34°C, typically around 32°C, and the pH is between pH 3 and 6, for example, around pH 4–5.

[0206] In addition to fermenting microorganisms (e.g., yeast), fermentation may also involve nutrients and additional enzymes, including phytase. The use of yeast in fermentation is well known in the art.

[0207] In other embodiments, as known in the art, the use of suitable fermenting microorganisms can produce fermentation end products, including, for example, glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lactic acid, amino acids, and derivatives thereof. More specifically, when lactic acid is the desired end product, species of the genus *Lactobacillus* (*Lactobacillus casei*) can be used. L. casei When glycerol or 1,3-propanediol is the desired end product, *Escherichia coli* can be used; and when 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired end products, *Ureaplasma citrinum* can be used as the fermentation microorganism. The list above is merely an example, and those skilled in the art will recognize many fermentation microorganisms that can be used to obtain the desired end product.

[0208] Method for producing fermentation products from materials containing ungelatinized starch This invention relates to a method for producing fermentation products from starch-containing materials without gelatinization (i.e., without cooking) (commonly referred to as the "crude starch hydrolysis" method). Fermentation products such as ethanol can be produced without liquefying an aqueous slurry containing the starch-containing material and water. In one embodiment, the method of the invention comprises saccharifying (e.g., milled) starch-containing material (e.g., granular starch) at a temperature below the initial gelatinization temperature, preferably in the presence of α-amylase and / or enzymes that produce carbohydrate sources, to produce a variety of sugars that can be fermented into fermentation products by suitable fermentation organisms. In this embodiment, the desired fermentation product, such as ethanol, is produced from ungelatinized (i.e., uncooked), preferably milled grains such as corn.

[0209] Therefore, in one aspect, the present invention relates to methods for producing fermentation products from starch-containing materials, the methods comprising: simultaneously saccharifying and fermenting the starch-containing material at a temperature below the initial gelatinization temperature of the starch-containing material using enzymes that produce a carbohydrate source and fermentation organisms. Saccharification and fermentation may also be separate processes. Thus, in another aspect, the present invention relates to methods for producing fermentation products, the methods comprising the following steps: (i) Saccharifying starch-containing materials at a temperature below the initial gelatinization temperature; and (ii) Fermentation using fermentation organisms; Step (i) is performed using at least one variant of glucosyl amylase as described in this invention.

[0210] In one embodiment, α-amylase is added in step (i). In another embodiment, steps (i) and (ii) are performed simultaneously.

[0211] In one embodiment, a protease is also present. The protease can be any acidic fungal protease or metalloproteinase. After fermentation, the fermentation products (e.g., ethanol) can optionally be recovered, for example, by distillation. Typically, one or more amylases such as glucosylamylase, and / or other carbohydrate-producing enzymes, and / or one or more α-amylases are present during fermentation. Examples of glucosylamylase and other carbohydrate-producing enzymes include glucosylamylases that hydrolyze the raw starch. Examples of one or more α-amylases include acidic α-amylases such as acidic fungal α-amylase. Examples of fermentation organisms include yeast, for example, strains of *Saccharomyces cerevisiae*. The term "initial gelatinization temperature" refers to the lowest temperature at which starch gelatinization occurs. Generally, starch heated in water begins to gelatinize between about 50°C and 75°C; the exact gelatinization temperature depends on the specific starch and can be readily determined by those skilled in the art. Therefore, the initial gelatinization temperature can vary depending on the plant species, the specific variety of the plant species, and the growing conditions. In the context of this invention, the initial gelatinization temperature of a given starch-containing material is the temperature at which 5% of the starch granules lose birefringence, as described by Gorinstein and Lii, 1992, Starch / Stärke [Starch] 44(12): 461-466. Prior to starting this method, a slurry of a starch-containing material (e.g., granular starch) having 10-55 w / w % dry solids (DS), preferably 25-45 w / w % dry solids, more preferably 30-40 w / w % dry solids can be prepared. This slurry may include water and / or process water, such as boil-distillate (countercurrent), scrubber water, evaporator condensate or distillate, side-stream stripper water obtained by distillation, or process water from other fermentation product equipment. Since the method of this invention is carried out below this initial gelatinization temperature and therefore no significant increase in viscosity occurs, high levels of boil-distillate can be used if desired. In one embodiment, the aqueous slurry comprises from about 1 to about 70 vol.%, preferably 15-60 vol.%, and especially from about 30 to 50 vol.%, water and / or process water, such as kettle distillate (countercurrent), scrubber water, evaporator condensate or distillate, water from a side-stream stripping column obtained by distillation, or process water from other fermentation product equipment, or combinations thereof. The starch-containing material can preferably be prepared by dry or wet milling to reduce the particle size to 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After undergoing the method of the invention, 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 preferably at least 99% of the dry solids in the starch-containing material are converted into soluble starch hydrolysate.The method in this aspect of the invention is carried out at a temperature below the initial gelatinization temperature, meaning the temperature is typically between 30°C and 75°C, preferably between 45°C and 60°C. In one embodiment, the method is carried out at a temperature between 25°C and 40°C, such as between 28°C and 35°C, such as between 30°C and 34°C, preferably about 32°C. In one embodiment, the method is performed to maintain sugar levels, such as glucose levels, at low levels, such as below 6 w / w%, such as below about 3 w / w%, such as below about 2 w / w%, such as below about 1 w / w%, such as below about 0.5 w / w%, or below 0.25 w / w%, such as below about 0.1 w / w%. Such low sugar levels can be achieved simply by using adjusted amounts of enzymes and fermentation organisms. Those skilled in the art can readily determine the dosage / quantity of the enzymes and fermentation organisms to be used. The amount of enzymes and fermentation organisms used can also be selected to maintain a low maltose concentration in the fermentation broth. For example, maltose levels can be maintained below about 0.5 w / w%, such as below about 0.2 w / w%. The method of the present invention can be carried out at a pH between about 3 and 7, preferably between pH 3.5 and 6, or more preferably between pH 4 and 5. In one embodiment, fermentation is carried out for 6 to 120 hours, particularly for 24 to 96 hours.

[0212] Method for producing fermentation products from materials containing gelatinized starch In this respect, the present invention relates to methods for producing fermentation products, particularly ethanol, from starch-containing materials, methods comprising: a liquefaction step, and saccharification and fermentation steps performed sequentially or simultaneously. Therefore, the present invention relates to methods for producing fermentation products from starch-containing materials, methods comprising the following steps: (a) Liquefying starch-containing materials in the presence of α-amylase; (b) Saccharifying the liquefied material obtained in step (a) using the glucoamylase of the present invention; (c) Fermentation using fermentation organisms; Steps (a) and / or (b) are performed in the presence of the glucosylamylase according to the present invention.

[0213] In one embodiment, a protease, such as an acidic fungal protease or a metalloproteinase, is added before, during, and / or after liquefaction. In one embodiment, the metalloproteinase is derived from a strain of *Thermoascus*, for example, *Thermoascus aurantiacus*, particularly *Thermoascus aurantiacus* CGMCC No. 0670. In another embodiment, the protease is a bacterial protease, particularly one derived from a strain of *Thermococcus*, and more particularly from *Thermococcus viridans* disclosed in US 6,358,726.

[0214] Additional glucosylamylase may be added. In one embodiment, the additional glucosylamylase is derived from strains of the genus *Aspergillus*, such as *Aspergillus niger* or *Aspergillus avocado*; strains of the genus *Arthrophyllus*, especially *Arthrophyllus emersonii*; or strains of the genus *Ataenia*, especially *Ataenia loureirii*; strains of the genus *Cordyceps*, such as *Cordyceps vulgaris*; strains of the genus *Entoloma*, such as *Entoloma spp.* or *Entoloma spp.*; or mixtures thereof. The saccharification step (b) and the fermentation step (c) may be performed sequentially or simultaneously. When the method is carried out as a sequential saccharification and fermentation process, pullulanase and / or protease may be added during saccharification and / or fermentation, and when steps (b) and (c) are performed simultaneously (SSF process), they are added before or during fermentation. Pullulanase and / or protease may also advantageously be added before liquefaction (pre-liquefaction treatment) (i.e., before or during step (a)) and / or after liquefaction (post-liquefaction treatment) (i.e., after step (a)). Pullulanase is most advantageously added before or during liquefaction (i.e., before or during step (a)). Fermentation products, such as, in particular, ethanol, can optionally be recovered after fermentation, for example, by distillation. The fermenting organism is preferably yeast, preferably a Saccharomyces cerevisiae strain. In a specific embodiment, the method of the invention further includes the following steps before step (a): x) Preferably, the particle size of the starch-containing material is reduced by grinding (e.g., using a hammer mill); y) to form a slurry containing the starch-containing material and water.

[0215] In one embodiment, the particle size is smaller than a #7 sieve, for example, a #6 sieve. A #7 sieve is commonly used in conventional prior art processes. The aqueous slurry may contain starch-containing material with a w / w% dry solids (DS) of 10-55, for example 25-45 and 30-40. The slurry is heated above the gelatinization temperature and an α-amylase variant may be added to initiate liquefaction (dilution). In one embodiment, the slurry may be spray-cooked prior to α-amylase treatment in step (a) to further gelatinize the slurry. In one embodiment, liquefaction may be performed as a three-step hot slurry method. The slurry is heated to between 60°C and 95°C, preferably between 70°C and 90°C, such as preferably between 80°C and 85°C, at pH 4-6, preferably 4.5-5.5, and optionally an α-amylase variant is added along with pullulanase and / or protease, preferably metalloproteinase, to initiate liquefaction (dilution). In one embodiment, the slurry can then be spray-cooked at a temperature between 95°C and 140°C, preferably 100°C and 135°C, such as 105°C and 125°C, for about 1-15 minutes, preferably about 3-10 minutes, and especially about 5 minutes. The slurry is cooled to 60°C and 95°C and additional α-amylase and optionally pullulanase and / or protease, preferably metalloproteinase, is added to complete the hydrolysis (secondary liquefaction). The liquefaction process is typically carried out at a pH of 4.0-6, particularly between 4.5 and 5.5. The saccharification step (b) can be performed using conditions well known in the art. For example, the entire saccharification process can last from about 24 to about 72 hours; however, typically only a pre-saccharification of 40-90 minutes is carried out at a temperature between 30°C and 65°C, typically at about 60°C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at temperatures ranging from 20°C to 75°C, preferably from 40°C to 70°C, typically around 60°C, and at a pH between 4 and 5, generally around pH 4.5. The most widely used method in the production of fermentation products, especially ethanol, is simultaneous saccharification and fermentation (SSF), in which there is no holding phase, meaning that the fermentation organism (such as yeast) and enzymes can be added together. SSF can typically be performed at temperatures ranging from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around 32°C. In one embodiment, fermentation takes place for 6 to 120 hours, particularly 24 to 96 hours.

[0216] Starch-containing materials Any suitable starch-containing starting material can be used in the methods of the present invention. The starting material is typically selected based on the desired fermentation product. Examples of suitable starch-containing starting materials for use in the methods of the present invention include barley, legumes, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains or any mixture thereof. The starch-containing material can also be waxy or non-waxy types of corn and barley. In a preferred embodiment, the starch-containing material is corn. In a preferred embodiment, the starch-containing material is wheat.

[0217] Fermentation products The term "fermentation product" refers to a product produced by a fermentation method or process that involves the use of fermenting organisms. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B vitamins). 12 (e.g., β-carotene); and hormones. In a preferred embodiment, the fermentation product is ethanol, such as fuel ethanol; drinking ethanol, i.e., neutral drinking alcohol; or industrial ethanol or products used in the consumer alcohol industry (e.g., beer and wine), the dairy industry (e.g., fermented dairy products), the leather industry, and the tobacco industry. Preferred beer types include ale, stout, porter, lager, bitters, malt liquor, happyhu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. In a preferred embodiment, the fermentation product is ethanol.

[0218] The invention is further described in the following numbered paragraphs: Paragraph [1]. A glucosyl amylase variant comprising substitutions at one or more positions selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, I13, K15, V18, L19, N25, S27, K28, S30, V36, V37, T43, D45, S57, V59, F60, I71, S73, T74, L77, D82, D83, V85, T86, E88, L91, S95, P97, T103, D114, S134, L137, T139, N142, L145, S146, N147, N149, Y152, V153, T154, S155, N156. L157, W158, P159, I160, Q162, V169, S170, S175, T176, Y177, D184, S186, R 199, A202, A203, T206, Q210, T211, S212, Q213, V214, S215, Y217, T218, T2 19. Q220, A221, D222, N223, L224, F227, Y231, P234, S235, Y238, T240, T24 3. G244, G245, G246, R247, S248, A252, T254, L255, Y262, S265, G267, A270, A271, K279, S282, L284, V294, Y295, S296, I297, N298, S299, G300, A302, S 303, N304, T309, E314, S316, Q318, G319, T326, V330, N339, E342, S343, E3 48. S351, T352, Q359, S362, G363, V364, T365, A366, S371, S372, T378, S38 1. I383, N385, F386, A392, N394, K396, Y408, K410, D412, S414, S417, V419, A426, S427, E433, A434, N436, N437, T438, Q439, G442, A446, L448, V450, N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N495, S501, A502, T506, I509, T510, N512, S516, A518, I519, N527, N528, A530, E534, D536, P537, N538, N539, I541, A545, S546, G547, S548, N552, and T554,The positions described therein correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more of these positions provide a glucosylamylase variant having an increase in melting temperature of at least 0.5 degrees Celsius as measured by TSA; and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0219] Paragraph [2]. According to the glucosylamylase variant described in paragraph 1, the variant contains substitutions at one or more positions selected from the group consisting of: Q1K, Q1R, S2E, S2K, S2L, S2P, S2R, V3L, V3G, V3R, D4R, D4S, D4G, D4A, D4W, S5L, S5V, S5G, S5C, S5R, S8Q, S8H, S8A, S8Y, S9C, S9Q, S9M, S9W, S9D, S9G, I13V, I13R, I13S, I13L, I13E, K15G, K15R, V18M, V18Q, L19G, L19F, N25S, N25A, S27A, S27L, S27G, S27V, S27C, K28C, K28R, S30Q, S30A, S30K, S30T, S30L, V36K, V36G, V36W, V3 6A, V36I, V37R, V37K, V37G, V37C, V37M, V37S, V37T, V37D, T43K, D45L, D45P , S57P, S57L, S57G, S57F, S57R, S57T, S57A, V59T, V59S, V59E, F60S, I71M, I 71S, I71T, I71V, S73H, S73R, S73N, S73V, S73G, T74V, L77S, L77P, L77R, D82 N, D82R, D82V, D82G, D83L, D83C, D83W, V85Q, V85G, V85P, T86R, T86V, E88Q , E88R, E88G, L91S, L91P, L91G, S95A, S95T, S95V, P97T, P97I, P97R, T103Y, T103A, T103G, D114G, D114N, D114M, D114R, D114C, S134P, S134A, S134V, S1 34W, S134D, S134H, S134L, S134G, L137W, L137S, L137A, L137V, L137G, L137 D. L137R, L137P, T139D, T139P, T139V, N142Y, N142H, N142C, L145C, L145D, L145G, L145V, L145S, S146W, S146L, S146R, S146G, S146P, N147Q, N147V, N1 47L, N147K, L147D, L147Y, L147H, L147S, N149H, N149T, N149R, N149K, N149 S, Y152S, Y152A, Y152R, Y152L, Y152K, Y152E, Y152P, Y152V, Y152I, Y152C,Y152W、V153E、V153S、V153G、V153W、V153Y、T154N、T154R、T154K、T154P、T154V、S155C、S155P、S155R、S155G、S155A、N156I、N156A、N156V、N156R、N156T、N156K、L157P、L157R、L157A、L157G、L157W、W158T、W158A、W158M、W158V、W158R、W158P、P159S、P159G、P159L、P159V、P159A、P159R、P159Q、P159E、I160A、I160G、I160N、I160T、I160R、I160V、Q162L、Q162V、Q162H、Q162P、Q162R、V169A、V169L、V169W、V169S、V169D、V169R、V169E、S170A、S170P、S170R、S170M、S175L、S175C、S175W、T176R、T176L、T176N、T176A、T176S、T176I、Y177H、D184P、D184W、D184S、D184Y、D184G、S186A、S186R、S186W、R199F、R199E、R199L、R199C、R199K、A202R、A202W、A202E、A202S、A202V、A203M、A203W、A203P、A203L、T206C、T206P、T206G、T206A、T206R、Q210C、Q210G、Q210S、Q210R、Q210L、Q210P、Q210V、T211R、T211A、T211H、T211K、T211Q、T211G、T211W、T211E、T211I、T211V、T211P、T211L、T211D、S212D、S212E、S212L、S212P、S212T、Q213W、Q213V、Q213D、Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、F227G、F227W、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、S235C、S235R、S235N、S235G、S235W、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、T243V、T243S、T243L、T243R、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、R247E、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W、S248K、S248T、A252E、A252T、A252Y、A252V、A252L、T254D、T254W、T254V、T254G、T254A、L255R、L255Q、L255P、L255G、Y262C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A270L、A270M、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R、S296F、S296L、S296W、S296K、I297S、I297P、I297K、I297F、I297R、I297W、N298W、N298G、N298C、N298V、N298L、N298A、S299P、S299C、S299M、S299L、S299T、G300S、G300A、G300P、G300L, G300W, A302G, A302L, A302C, A302R, A302V, S303P, S303V, S303C, S303A, S303R, N304T, N304R, N304Q, N304L, N304V, T309G, T309I, T309R, T309 M、E314Y、E314T、E314V、E314G、E314S、E314L、E314A、S316T、S316L、S316G、 S316F、S316R、S316P、S316V、S316Q、Q318L、Q318R、G319R、G319Q、G319P、G31 9A, T326V, T326G, T326W, T326N, T326A, V330S, V330L, V330P, V330R, V330A, V330G, N339P, N339A, N339T, E342M, E342W, E342N, E342L, E342R, S343R, S 343C, E348W, E348F, E348P, E348V, E348G, E348M, S351P, S351C, S351G, S351R, S351L, S351W, T352P, T352L, T352G, T352Q, T352Y, Q359K, Q359P, Q359R Q359S, Q359A, S362P, S262R, S262G, S262M, G363R, G363T, G363P, V364A, V364C, V364E, V364S, V364G, V364L, T365S, T365G, T365W, T365L, T365H, A366 D, A366T, A366P, A366R, A366H, S371A, S371G, S372A, S372E, S372C, S372L, S372R, T378G, T378L, T378D, T378H, T378A, T378P, S381K, I383A, I383G, I38 3C、I383L、I383T、I383M、N385R、N385W、N385S、N385G、N385D、F386S、F386W 、F386Q、F386V、F386I、F386G、F386C、F386A、F386T、F386L、A392V、A392L、A 392E、A392G、N394D、N394R、N394Y、N394W、N394E、K396I、K396W、K396P、K39 6Y、K396F、Y408V、Y408E、Y408P、Y408S、Y408K、Y408L、K410S、K410R、D412M、D412S、D412N、D412W、D412L、D412R、S414C、S414R、S414G、S414V、S414W、S414H、S417Y、V419S、V419G、V419C、V419A、V419K、V419R、V419T、A426M、A426N、A426K、A426R、S427G、S427A、S427P、S427N、S427D、S427L、E433C、A434Q、A434G、N436S、N436P、N436D、N437K、N437R、N437T、N437P、T438E、T438G、Q439W、Q439S、Q439G、Q439C、Q439R、Q439Y、G442V、G442D、G442C、G442A、G442L、G442W、G442E、G442M、G442R、A446G、A446D、A446R、A446E、A446I、L448G、L448P、L448E、V450P、V450S、V450C、V450E、V450L、V450N、N470H、N470D、N470K、N470V、N470L、E472I、V474W、V474C、V474A、V474L、V474G、W475P、W475A、W475R、N478L、N478I、N478P、N478R、N478W、N478S、N478G、N478K、N478A、S484G、S484Y、S484P、S484A、S484N、V485A、V485W、V485K、V485G、V485R、D486I、D486K、D486Y、D486S、D486A、D486W、D486L、A487S、A487V、A487L、A487G、A487C、A487K、S492L、S492R、S492T、S492W、S492P、S492C、A493V、A493R、A493D、A493W、D494N、D494R、D494G、D494L、D494E、D494Q、N495L、N495W、N495G、N495R、N495C、S501R、S501L、S501M、S501K、S501W、A502C、A502Q、A502W、A502G、A502V、T506A、T506P、T506V、I509E、I509D、I509S、I509F、I509W、I509R、T510F、T510E、T510R、T510P、T510V、T510A、T510L、N512Q、N512K、N512H、N512R, N512V, S516R, S516W, S516P, S516K, S516Y, S516C, A518D, A518G, A518Y, A518V, A5 18R, A518L, A518T, I519W, I519L, I519R, I519F, I519K, N527T, N527K, N527P, N527L, N528 D. N528G, N528K, N528V, N528E, N528L, A530R, A530C, A530G, A530V, A530S, A530T, E534W, E534Q, E534C, E534V, E534G, E534R, E534F, E534K, D536G, D536R, D536W, D536H, D536K, D53 6N, D536M, D536C, D536V, P537D, P537M, P537W, P537G, P537E, N538D, N538S, N538W, N538Y , N538A, N539M, N539R, N539P, N539A, I541A, I541T, I541V, I541G, I541N, A545R, A545T, A5 45V, A545L, S546P, S546G, S546C, S546E, S546N, G547D, G547S, G547V, S548P, S548W, S548L, S548G, S548T, N552V, N552E, N552F, N552A, N552R, N552G, and T554Q, T554G, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in melting temperature of at least 0.5 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0220] Paragraph [3]. According to the glucosylamylase variants described in paragraphs 1-2, the variants contain substitutions at one or more positions selected from the group consisting of: S2, V3, D4, S8, S9, I13, V18, L19, S27, K28, S30, V36, V37, T43, S57, V59, S73, T74, L77, D82, V85, T86, L91, S95, P97, D114, S134, L137, T139, N142, L145, S146, N147, N149, Y152, V153, T154, S155, N156, L157, W1 58. P159, I160, Q162, S170, S175, T176, Y177, D184, S186, R199, A203, T206, Q210, T211, S212, Q213, V214, S215, Y217, T218, T219, Q22 0. A221, D222, N223, L224, F227, Y231, P234, S235, Y238, T240, G244, G245, G246, S248, A252, T254, L255, Y262, S265, G267, A271, K279 , S282, L284, V294, Y295, S296, I297, N298, S299, G300, S303, N304, T309, E314, S316, Q318, G319, T326, V330, N339, E342, S343, E348, T352, Q359, G363, V364, A366, S371, S372, T378, S381, I383, N385, F386, A392, N394, K396, Y408, K410, D412, S414, S417, V419, A426, S 427, A434, N436, T438, Q439, G442, L448, V450, N470, E472, V474, W475, S484, V485, D486, A487, S492, A493, D494, N495, A502, T506, I509, T510, N512, S516, A518, I519, N527, N528, A530, E534, P537, N538, N539, I541, A545, S546, G547, S548, N552, and T554, wherein the positions correspond to SEQ The amino acid positions in the amino acid sequence shown in ID NO: 3; and the substitutions at one or more of the positions provided by the substitution provide a glucosylamylase variant having at least 0 as measured by TSA.The melting temperature increases by 7 degrees Celsius, and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence similarity to the amino acid sequence having SEQ ID NO: 3.

[0221] Paragraph [4]. According to the glucosylamylase variants described in paragraphs 1-3, the variants contain substitutions at one or more positions selected from the group consisting of: S2, S8, V18, K28, V36, V37, T43, S57, V59, S73, T74, T86, S95, P97, D114, S134, L137, T139, N142, L145, S146, N147, N149, Y152, V153, T154, S155, L1 57. W158, P159, I160, Q162, S170, S175, T176, Y177, D184, S186, R199, A203, T206, Q210, T211, S212, Q2 13. V214, S215, Y217, T218, T219, Q220, A221, D222, N223, L224, F227, Y231, P234, Y238, T240, G244, G2 45. G246, S248, A252, T254, L255, Y262, S265, G267, A271, K279, S282, L284, V294, Y295, S296, I297, N2 98. G300, S303, N304, T309, E314, Q318, T326, V330, E342, S343, E348, T352, Q359, S371, T378, I383, F3 86, N394, K396, Y408, K410, D412, S417, V419, A426, S427, A434, Q439, G442, L448, N470, E472, W475, V485, D486, S492, A493, D494, N495, I509, T510, I519, N527, P537, N538, S546, G547, and T554, wherein the positions correspond to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitution at one or more of the positions provides a glucosylamylase variant having an increase in melting temperature of at least 0.9 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0222] Paragraph [5]. According to the glucosyl amylase variants described in paragraphs 1-4, the variants contain substitutions at one or more positions selected from the group consisting of: V18, K28, V36, V37, T43, S57, V59, S73, T86, S95, T139, N142, L145, S146, N147, N149, Y152, V153, T154, L157, W158, P159, S170, S175, T176, D184, S186, R199, A203, Q210, T211, Q213, V2 14. S215, Y217, T218, T219, Q220, A221, D222, N223, L224, Y231, P234, Y238, T240 , G244, G245, G246, S248, A252, T254, L255, Y262, S265, G267, A271, K279, S282, L 284, V294, Y295, S296, I297, N298, G300, S303, N304, T309, E314, T326, E342, S34 3. T352, Q359, I383, K396, K410, D412, S417, V419, A426, S427, A434, Q439, N470, W475, D486, S492, A493, D494, N495, T510, P537, N538, G547, and T554, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitution at one or more positions provides a glucosylamylase variant having an increase in melting temperature of at least 1.1 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0223] Paragraph [6]. According to the glucosylamylase variants described in paragraphs 1-5, the variants contain substitutions at one or more positions selected from the group consisting of: K28, T43, S57, S73, S95, T139, L145, N149, Y152, V153, L157, W158, P159, S175, D184, S186, R199, A203, Q210, T211, Q213, V214, Y217, T218, T219, A221, D2 22, L224, Y231, P234, Y238, T240, G244, G246, S248, A252, T254, L255, Y262, A271, K279, L284, V294, Y295, S296, I297, N298, N304, S343, T352, K410, D412, V419, S427, N470, W475, S492, A493, and P537, wherein the positions correspond to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitution at one or more of the positions provides a glucosylamylase variant having an increase in melting temperature of at least 1.5 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence of SEQ ID NO: 3.

[0224] Paragraph [7]. According to the glucosylamylase variants described in paragraphs 1-6, the variants contain substitutions at one or more positions selected from the group consisting of: K28, T43, S57, S95, T139, L145, N149, V153, L157, P159, S186, R199, A203, T211, V214, Y217, Y231, P234, Y238, T240, G244, G246, S248, T254, L255, A271, L284, V294, Y295, S296, K410, S492, and A493, wherein the positions correspond to in SEQ ID The amino acid positions in the amino acid sequence shown in NO:3; and the substitutions at one or more of the positions provide a glucosylamylase variant having an increase in melting temperature of at least 2.0 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence having SEQ ID NO:3.

[0225] Paragraph [8]. A glucosyl amylase variant comprising substitutions at one or more positions selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, G11, I13, K15, A16, V18, L19, N25, S27, S30, A32, A34, V36, V37, S44, S57, V59, F60, Y67, T68, I71, D72, S73, T74, S75, S76, L77, R78, D82, D83, F84, V85, T86, N90, L91, Q93, S95, L101, T102, T103, S134. L137, T139, N142, L145, S146, N147, Y152, V153, T154, S155, L157, W158, P159, I160, Q162, N163, S170, S175, T176, Y177, S186, R199, A20 2. A203, T206, Q210, T211, S212, Q213, S215, Q220, A221, D222, N223, L224, F227, P234, S235, Y238, T240, T243, G244, G245, G246, S248, A 252, T254, L255, A270, A271, K279, S282, L284, Y295, S296, I297, N298, S299, G300, A302, S303, N304, S316, G319, T326, V330, N339, E342 , S343, Q344, E348, S351, Q359, S362, G363, T365, A366, S371, S372, T378, S381, I383, F386, A392, N394, K396, N401, K410, D412, S414, S4 17, V419, D420, E433, N437, T438, Q439, F440, G442, A446, N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N495, S501, A502, I509, T510, N512, S516, A518, I519, N527, A530, E534, P537, N538, N539, I541, A545, S546, G547, N552, and T554, wherein the positions correspond to SEQ The amino acid positions in the amino acid sequence shown in ID NO: 3; and the substitutions at one or more of the positions provided a glucosylamylase variant having a value of at least 1 as measured as an improvement factor IF.The specific activity of 1 is increased, and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0226] Paragraph [9]. The glucosyl amylase variants described in paragraph 8, which contain substitutions at one or more positions selected from the group consisting of: Q1R, Q1L, Q1T, Q1G, Q1P, Q1K, Q1M, Q1F, Q1S, Q1A, Q1W, S2V, S2Q, S2E, S2D, S2P, S2A, S2T, S2L, S2R, S2K, S2W, S2G, V3G, V3L, V3I, V3A, V3E, D4R, D4C, D4S, D4G, D4N, D4V, D4W, D4F, D4A, S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S8A, S8W, S8R, S8L, S8 Y, S8G, S8M, S8H, S8P, S8Q, S8V, S8C, S8E, S8K, S8T, S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, G11D, I13L, I13A, I13Q, I13S, I13D, I13R, I13 M, I13V, I13G, I13Y, I13E, K15V, K15R, K15I, K15M, K15A, K15F, K15L, K15S, K15E, K15W, K15G, K15D, A16L, A16V, A16G, A16E, A16S, A16T, A16K, A16G, V18 A. V18R, V18M, V18T, V18L, V18Q, V18I, L19S, L19A, L19K, L19V, L19C, L19H, L19W, L19F, L19R, N25W, N25Y, N25D, N25F, N25G, N25R, N25V, N25L, N25A, N2 5S, N25E, N25C, N25Q, S27A, S27W, S27H, S27V, S27T, S27C, S27G, S27E, S27L , S27F, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S30T, S30V, A32D, A 32E, A32S, A32V, A32R, A32G, A32M, A32T, A32C, A32K, A32W, A34W, A34R, A34 L, A34Q, A34G, A34C, A34F, A34V, A34E, A34T, A34I, A34P, V36I, V36R, V36A, V36G, V36L, V37C, V37G, V37R, V37A, V37M, S44R, S44W, S44L, S44T, S44C, S4 4A, S44V, S44P, S44E, S57G, S57T, S57H, S57P, S57A, V59T, V59E, V59Q, V59L,V59R、、F60S、F60V、F60A、F60I、Y67C、Y67N、Y67A、Y67G、Y67T、Y67V、Y67D、Y67H、Y67R、Y67F、Y67L、Y67P、Y67S、Y67M、T68K、T68C、T68A、T68P、T68R、T68Q、I71T、I71M、I71V、I71S、I71N、I71F、I71D、I71P、I71R、I71L、I71K、D72L、D72G、D72N、D72R、D72K、D72E、D72W、D72A、D72C、D72Y、D72S、D72Q、D72T、S73H、S73G、S73N、S73C、S73R、S73V、S73L、S73I、S73W、S73P、T74S、T74E、T74P、T74N、T74F、T74P、T74M、T74R、T74C、S75G、S75N、S75P、S75E、S75C、S75R、S75L、S75K、S75I、S75T、S76H、S76P、S76Q、S76E、L77S、L77Y、L77E、L77P、R78W、R78G、R78K、R78Q、R78T、R78A、R78C、R78M、R78E、D82V、D82G、D82R、D82N、D82E、D82C、D83L、D83C、D83W、D83A、D83R、D83G、D83V、D83S、D83E、F84Y、F84L、F84S、F84T、F84P、F84E、F84V、F84A、F84W、F84K、F84M、F84R、V85G、V85W、V85P、V85Q、V85E、V85H、V85R、V85T、T86C、T86R、T86G、T86W、T86D、T86V、T86S、T86A、N90G、N90E、N90T、N90P、N90C、L91H、L91P、L91F、L91V、L91R、Q93L、Q93M、Q93C、Q93H、Q93G、Q93R、Q93W、Q93D、Q93A、Q93N、Q93K、S95V、S95R、S95D、S95Y、S95G、S95Q、S95A、S95K、L101M、L101V、L101R、L101P、L101F、L101H、L101A、L101G、L101N、L101K、L101C、T102N、T102S、T102C、T102R、T102A、T102I、T102M、T102W、T102E、T102P、T102F、T103A、T103S、T103G、T103D、T103I、T103E、T103V、T103N、S134V、S134I、S134M、S134P、S134L、S134A、S134C、L137S、L137D、L137W、L137G、L137R、L137A、L137I、L137T、T139A、T139N、T139S、T139G、T139D、T139H、T139R、N142K、N142E、N142Q、N142R、N142G、N142H、N142W、N142A、L145S、L145W、L145N、L145C、L145V、L145R、L145D、S146V、S146G、S146L、S146T、S146A、S146C、S146P、S146F、S146R、S146W、N147K、N147E、N147S、N147F、N147T、N147I、N147D、N147P、N147Y、N147H、N147L、Y152V、Y152E、Y152L、Y152I、Y152A、Y152M、Y152R、Y152F、Y152G、V153R、V153Y、V153C、T154R、T154G、T154L、T154S、T154A、T154M、T154P、S155R、S155G、S155L、S155A、S155H、S155W、S155C、S155I、S155P、S155M、S155N、S155T、L157P、L157Q、L157V、L157M、L157R、W158R、W158E、W158C、W158K、W158L、W158G、P159S、P159R、P159V、P159Q、P159T、P159D、P159A、P159L、P159G、I160T、I160A、I160V、I160D、I160G、I160S、I160L、I160Y、I160N、I160F、Q162L、Q162K、Q162R、Q162S、Q162H、Q162P、Q162I、Q162V、N163D、N163G、N163R、N163T、N163I、N163Q、N163Y、N163K、N163H、N163W、N163A、N163S、S170A、S175W、S175R、S175T、S175C、T176S、T176R、T176L、T176A、T176W、T176I、Y177S、Y177T、Y177D、Y177V、S186V、S186R、S186E、S186L、S186D、S186C、S186A、S186Q、R199K、R199V、R199A、R199M、R199N、R199W、R199T、R199E、A202S、A202T、A202Q、A202L、A202E、A202P、A202V、A202F、A202W、A202G、A203Q、A203K、A203W、A203R、A203V、A203L、A203M、A203T、A203E、A203G、A203S、A203P、T206I、T206S、T206W、T206V、T206A、T206P、T206G、T206R、Q210D、Q210R、Q210G、Q210A、Q210L、Q210H、Q210P、Q210V、Q210I、Q210C、T211P、T211R、T211S、T211D、T211Q、T211H、T211A、T211L、T211G、T211W、S212V、S212K、S212D、S212T、S212H、S212L、S212P、S212E、S212C、S212A、S212M、Q213Y、Q213D、Q213R、Q213N、Q213S、Q213W、Q213K、Q213L、Q213C、Q213P、S215L、S215T、S215Q、S215R、S215V、S215G、S215N、S215C、Q220L、Q220P、Q220K、Q220R、Q220H、Q220E、A221V、A221T、A221E、A221G、A221P、D222E、D222M、D222A、D222G、D222N、D222V、D222H、N223K、N223R、L224V、F227A、F227V、F227L、F227S、F227Y、F227E、F227G、P234A、P234L、P234Q、P234S、S235C、S235R、S235W、S235G、S235K、Y238C、Y238L、Y238E、Y238W、Y238A、Y238S、Y238G、T240L、T240C、T240G、T240W、T240V、T240R、T240S、T240A、T240E、T243S、T243Q、T243M、T243G、T243L、T243V、T243E、T243P、T243R、T243W、G244W、G244D、G244Y、G244A、G244S、G244R、G245M、G245N、G245S、G245T、G245V、G245D、G245I、G246V、G246W、G246M、G246E、G246N、G246Q、G246S、G246D、G246R、S248E、S248L、S248C、S248G、S248P、S2 48F、S248T、A252S、A252T、A252V、A252P、A252G、T254A、T254S、T254G、T254 P、L255V、L255A、L255P、L255I、L255C、A270W、A270T、A270E、A270C、A270M、 A270S、A270L、A270G、A270R、A270Y、A270V、A271R、A271P、A271L、A271W、A27 1G, A271T, K279V, K279W, K279A, K279L, K279R, K279E, K279Y, K279P, K279G, K279S, S282G, S282T, S282L, S282V, S282F, S282R, S282A, S282I, S282W, L 284V, L284G, L284S, L284M, L284T, Y295K, Y295H, Y295Q, Y295W, Y295M, Y295F, Y295C, Y295E, Y295V, S296A, S296T, S296K, S296N, S296Y, S296F, S296Q S296P, S296L, S296D, I297L, I297V, I297H, I297R, I297W, I297K, I297T, I297F, I297G, I297Q, N298M, N298D, N298S, N298R, N298K, N298A, N298V, N298 E、N298G、N298L、S299L、S299G、S299V、S299A、S299R、S299Q、S299M、S299I、 S299P、S299T、G300A、G300N、G300D、G300R、G300L、G300F、G300C、G300P、G30 0W, G300T, G300S, A302L, A302R, A302P, A302V, A302K, A302M, A302Y, A302S, A302T, A302G, S303P, S303K, S303R, S303C, S303A, S303F, S303W, S303L, S 303Q, N304V, N304G, N304P, N304W, N304F, N304E, N304T, N304D, N304R, N304S, N304A, N304I, N304M, N304K, S316T, S316C, S316A, S316R, S316P, S316HS316K, S316F, S316G, S316Q, S316N, S316M, S316L, S316V, G319T, G319R, G319W, G319S, G319Q, G319A, G319D, T326S, T326G, T326A, T326C, T326Y, T326 P、T326I、T326E、T326Q、V330M、V330G、V330I、V330D、V330P、V330L、V330Y、 V330S、V330A、N339T、N339R、N339S、N339A、N339Q、N339P、E342L、E342K、E34 2T, E342M, E342R, E342V, E342H, E342G, E342Q, E342S, E342F, E342A, E342W, S343A, S343W, S343G, S343P, S343Q, S343T, S343E, S343R, S343L, Q344L, Q 344V、Q344T、Q344D、Q344A、Q344H、Q344K、Q344R、Q344P、Q344E、E348C、E34 8G、E348V、E348M、E348N、E348A、E348I、E348D、E348L、E348K、E348R、S351Y、 S351G, S351R, S351C, S351N, S351L, S351K, S351V, S351F, S351T, S351A, S351P, S351W, Q359A, Q359V, Q359T, Q359R, Q359G, Q359L, Q359K, Q359S, Q359 P, Q359W, S362V, S362P, S362R, S362G, S362H, S362E, S362M, S362D, S362Y, S362C, S362F, S362A, S362Q, G363C, G363H, G363D, G363W, G363R, G363Q, G36 3S, G363A, G363T, G363P, T365R, T365W, T365G, T365L, T365C, T365Q, T365I, T365V, T365Y, T365S, T365E, A366R, A366L, A366I, A366Q, A366P, A366T, A 366S, A366E, A366G, A366D, A366W, A366H, S371V, S371R, S371A, S371T, S371G, S371C, S371E, S371P, S372P, S372E, S372R, S372A, S372Q, S372N, S372GS372R, S372L, S372V, S372M, S372C, S372W, T378P, T378A, T378K, T378W, T378M, T378Q, T378G, T378V, T378E, T378S, T378R, T378L, T378C, T378I, T378 D、S381E、S381Y、S381D、S381N、S381R、S381G、S381V、S381A、S381T、S381P、 S381W、S381Q、S381C、S381I、I383F、I383N、I383G、I383C、I383E、I383L、I38 3M, I383V, I383A, I383T, I383R, I383S, F386L, F386Y, F386R, F386S, F386G, F386M, F386C, F386W, F386A, A392V, A392R, A392T, A392S, A392E, A392L, A 392G, A392P, A392F, A392M, A392I, A392Q, N394A, N394S, N394T, N394R, N394H, N394G, N394C, N394E, N394W, N394P, N394L, N394V, N394F, N394Q, N394K K396S, K396P, K396M, K396F, K396Q, K396E, K396D, K396W, K396L, K396A, K396I, K396R, K396G, K396C, K396V, N401Q, N401V, N401F, N401S, N401T, N401 G、N401R、N401C、N401A、N401D、N401K、N401E、N401Y、N401W、N401P、N401L、 K410S、K410T、K410L、K410D、K410M、K410V、K410P、K410N、K410C、K410G、K41 0E、K410W、K410R、D412R、D412Q、D412S、D412P、D412E、D412N、D412G、D412V 、D412L、D412W、D412A、D412K、D412M、D412T、S414P、S414A、S414W、S414G、S 414L、S414R、S414E、S414N、S414T、S414Q、S417R、S417G、S417K、S417Y、S41 7A、S417N、V419D、V419E、V419A、V419G、V419M、V419L、V419I、D420V、D420A、E433W、E433P、E433M、E433Y、E433S、E433C、E433G、E433A、E433R、E433Q、E433K、N437V、N437E、N437D、N437M、N437T、N437A、N437S、N437W、N437L、N437P、N437Y、N437G、N437Q、N437K、N437R、T438R、T438A、T438K、T438W、Q439A、Q439R、Q439G、Q439W、Q439P、Q439C、Q439M、Q439Y、Q439D、F440T、F440L、F440W、F440E、F440S、G442V、G442L、G442D、G442A、G442C、G442S、G442F、G442M、G442I、G442Y、G442W、A446L、A446R、A446F、A446G、A446S、A446M、A446Q、A446W、A446V、A446P、A446D、N470W、N470G、N470L、N470S、N470P、N470Y、N470A、N470E、N470D、N470H、N470K、N470T、N470M、E472W、E472S、E472L、E472G、E472R、E472P、E472V、E472T、E472K、V474R、V474F、V474Y、V474I、V474M、V474W、V474E、V474Q、V474L、V474G、V474A、V474K、V474T、V474H、W475P、W475S、W475L、W475C、W475Q、W475G、W475R、W475T、N478V、N478A、N478S、N478T、N478R、N478K、N478G、N478L、N478M、N478I、N478D、N478W、N478E、S484Q、S484T、S484E、S484F、S484A、S484G、S484D、S484L、S484W、S484V、S484R、S484Y、S484P、S484M、V485L、V485T、V485A、V485S、V485R、V485G、V485I、V485E、V485D、V485F、V485K、D486I、D486G、D486R、D486E、D486S、D486A、D486T、D486K、D486F、D486M、D486Q、D486C、D486L、D486Y、D486P、A487M、A487E、A487V、A487S、A487C、A487G、S492L、S492P、S492V、S492R、S492Y、S492M、S492H、S492T、S492K、S492W、A493G、A493S、A493Y、A493V、A493T、A493E、A493Q、A493R、D494A、D494S、D494E、D494Q、D494Y、D494G、D494R、D494T、D494W、D494N、D494H、D494L、D494M、D494V、D494P、N495S、N495L、N495F、N495C、N495W、N495R、N495G、S501P、S501T、S501L、S501G、S501M、S501R、S501K、S501V、S501E、S501A、S501C、A502W、A502V、A502S、A502G、A502D、A502E、A502T、A502M、A502Y、A502H、I509G、I509R、I509W、I509A、I509V、I509L、I509S、I509P、I509T、I509E、I509H、I509N、T510R、T510I、T510A、T510H、T510S、T510Y、T510V、T510L、T510K、T510E、T510P、T510F、T510M、N512S、N512Q、N512L、N512G、N512W、N512I、N512M、N512Y、N512K、N512V、N512H、N512F、N512T、N512R、N512D、S516Y、S516R、S516P、S516T、S516G、S516V、S516N、S516L、S516F、S516M、S516A、S516W、S516C、S516K、A518G、A518P、A518W、A518V、A518R、A518L、A518M、A518F、A518Y、A518S、I519L、I519C、I519G、I519W、I519S、I519Y、I519N、I519A、I519V、I519Q、I519T、I519H、I519M、N527S、N527L、N527V、N527G、N527W、N527H、N527R、N527K、A530R、A530C、A530S、A530G、A530F、A530Y、A530W、A530T、A530V、E534M、E534A、E534V、E534W、E534C、E534R、E534T、E534L、E534G, E534F, E534S, E534Q, E534K, P537R, P537T, P537H, P537M, P537G, P537A, P537S, P537E, P537Y, P537L, P537V, N538G, N53 8V, N538R, N538A, N538W, N538D, N538M, N538S, N538I, N538Y, N539L, N539S, N539A, N539I, N539V, I541A, I541G, I541T, I541W, I541K, I541V, I541N, I541F, A545L, A545W, A545V, A545S, A545G, A545R, A545T, A545P, S546E, S546C, S546G, S546N, S546V, G547S, G547V, G547L, G547D, G547R, G547C, G547M, N552V, N552E, N552D, N552G, and T554A, T554G, T554E, T554D, T554C, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitutions at one or more positions thereon provide a glucosylamylase variant having an increase in specific activity of at least 1.1 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0227] Paragraph

[10] . Variations as described in paragraphs 8-9, which include substitutions at one or more locations selected from the group consisting of: Q1, V3, D4, S5, S8, S9, I13, A16, V18, S27, S30, A32, V37, S44, S57, V59, F60, Y67, I71, S73, T74, S75, L77, R78, F84, V85, T86, Q93, S95, L101, T102, T103, L137, N142, N147, Y152, V153, L157, W158, N163, S186, R1 99, Q210, T211, S212, S215, N223, L224, P234, S235, T240, T243, T254, K279, S282, I297, N298, S299, G300, S303, E342, S343, Q344, E348, G363, A366, S381, A392, Q439, V474, W475, D486, A493, A502, T510, A518, I519, A530, E534, N538, S546, and G547, wherein the positions correspond to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitution at one or more of the positions provides a glucosylamylase variant having an increase in specific activity of at least 1.2 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

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[11] . The variants described in paragraphs 8-10, comprising substitutions at one or more positions selected from the group consisting of: Q1, S5, S9, I13, V18, S30, V37, V59, I71, S73, T74, F84, V85, Q93, L137, N142, W158, N163, S186, S215, N223, P234, S235, S299, E348, Q439, D486, A493, E534, and S546, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in specific activity of at least 1.3 as measured by the improvement factor IF, and further wherein the variant is associated with the amino acid sequence shown in SEQ ID NO: 3. The amino acid sequence of 3 has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

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[12] . The variant as described in paragraphs 8-11, comprising substitutions at one or more positions selected from the group consisting of: S5, S9, S30, V37, S73, F84, V85, S186, S215, Q439, D486, E534, and S546, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant having an increase in specific activity of at least 1.5 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

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[13] . Variations as described in any of paragraphs 8-12, which include substitutions at one or more locations selected from the group consisting of: S5V, S5R, S5P, S5L, S5G, S5C, S5N, S5Q, S5T, S9D, S9Q, S9R, S9G, S9A, S9N, S9E, S9K, S9L, S9T, S9M, S30A, S30P, S30K, S30R, S30Q, S30Y, S30E, S30D, S30T, S30V, V 37C, V37G, V37R, V37A, V37M, S73A, S73H, S73G, S73N, S73C, S73R, S73V, S73L, S73I, S73W, S73P, F84Y, F84L, F 84S, F84T, F84P, F84E, F84V, F84A, F84W, F84K, F84M, F84R, V85G, V85W, V85P, V85Q, V85E, V85H, V85R, V85T, S 186V, S186R, S186E, S186L, S186D, S186C, S186A, S186Q, S215L, S215T, S215Q, S215R, S215V, S215G, S215N, S 215C, Q439A, Q439R, Q439G, Q439W, Q439P, Q439C, Q439M, Q439Y, Q439D, D486I, D486G, D486R, D486E, D486S, D 486A, D486T, D486K, D486F, D486M, D486Q, D486C, D486L, D486Y, D486P, E534M, E534A, E534V, E534W, E534C, E534R, E534T, E534L, E534G, E534F, E534S, E534Q, E534K, S546E, S546C, S546G, S546N, and S546V, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and the substitution at one or more of the positions provides a glucosylamylase variant having an increase in specific activity of at least 1.5 as an improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO: 3.

[0231] Paragraph

[14] . A variant as described in any of paragraphs 1-7, comprising a substitution corresponding to position 43, specifically T43K and wherein the increase in melting temperature compared to the melting temperature of the glucosyl amylase of SEQ ID NO: 3 is at least 2°C, for example at least 3°C.

[0232] Paragraph

[15] . A variant as described in any of paragraphs 1-13, comprising a substitution corresponding to a position selected from the group consisting of positions 4, 5, 13, 15, 18, 85, specifically substitutions selected from D4R, S5V, I13S, K15R, V18M, V85G, wherein the variant has improved specific activity compared to the glucosylamylase of SEQ ID NO: 3.

[0233] Paragraph

[16] . Variations as described in any of paragraphs 1-15, wherein the variations further include substitutions corresponding to S95P and A121P, especially S95P + A121P.

[0234] Paragraph

[17] . Variations as described in any of paragraphs 1-16, wherein the variations further include substitutions corresponding to S95P + A121P + Y295W or S95P + A121P + Y295W + Q318Y.

[0235] Paragraph

[18] . Variations as described in any of the preceding paragraphs, wherein the variation comprises at least one of the following substitutions or combinations of substitutions: T43K; D4R; S5V; I13S; K15R; V18M; V85G; S95P + A121P + Y295W + T43K; T43K + S95P + A121P + Y295W + Q318Y; V18M + T43K + S95P + A121P + Y295W + Q318Y; D4R + T43K + S95P + A121P + Y295W + Q318Y; S5V + T43K + S95P + A121P + Y295W + Q318Y; I13S + T43K + S95P + A121P + Y295W + Q318Y; V18M + T43K + S95P + A121P + Y295W; The variant is said to have glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 3, and the variant has increased specific activity and / or an increase in melting temperature of at least 2°C, specifically at least 3°C, as measured by TSA, compared with the glucosylamylase of SEQ ID NO: 3.

[0236] Paragraph

[19] . The variant described in any of paragraphs 1-18 has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence similarity to the amino acid sequence of SEQ ID NO: 3.

[0237] Paragraph

[20] . Variations as described in any of paragraphs 1-19, wherein the number of changes is 1-20, such as 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes.

[0238] Paragraph

[21] . A composition comprising a glucose amylase variant as described in any of paragraphs 1-19.

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[22] . The composition according to paragraph 21 further comprises pullulanase.

[0240] Paragraph

[23] . The composition according to paragraph 21 further comprises α-amylase.

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[24] . Use of the polypeptides described in any of paragraphs 1-19 for the production of syrups and / or fermentation products.

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[25] . A method for producing a fermentation product from a starch-containing material, the method comprising the steps of: (a) liquefying the starch-containing material in the presence of an α-amylase; (b) saccharifying the liquefied material; and (c) fermenting with a fermentation organism; wherein step (b) is performed using at least one variant of glucosylamylase as described in any of paragraphs 1-19.

[0243] Paragraph

[26] . According to the method described in paragraph 25, steps (b) and (c) are performed simultaneously.

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[27] . A method for producing fermentation products from starch-containing materials, the method comprising the following steps: (a) Saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of the starch-containing material; and (b) Fermentation using fermentation organisms. Step (a) is performed using at least one variant of glucosyl amylase as described in any of paragraphs 1-19.

[0245] Paragraph

[28] . A method for producing a syrup product from a starch-containing material, the method comprising the steps of: (a) liquefying the starch-containing material in the presence of an α-amylase; and (b) saccharifying the liquefied material in the presence of a variant glucosylase as described in any of paragraphs 1-19.

[0246] Paragraph

[29] . A method for producing a syrup product from a starch-containing material, the method comprising the steps of saccharifying the starch-containing material in the presence of a variant glucosylamylase as described in any of paragraphs 1-19 at a temperature below the initial gelatinization temperature of the starch-containing material.

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[30] . A polynucleotide that encodes a variant as described in any of paragraphs 1-19.

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[31] . A nucleic acid construct comprising the polynucleotides as described in paragraph 30.

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[32] . An expression vector comprising polynucleotides as described in paragraph 30.

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[33] . A host cell comprising a polynucleotide as described in paragraph 30, or a nucleic acid construct as described in paragraph 31, or an expression vector as described in paragraph 32.

[0251] Paragraph

[34] . The host cell described in paragraph 33 is a yeast cell, particularly of the genus *Saccharomyces*, such as *Saccharomyces cerevisiae*.

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[35] . The method described in any of paragraphs 25-27, wherein the host cell described in paragraph 34 is used as the fermentation organism in the fermentation step and the fermentation product is ethanol.

[0253] Paragraph

[36] . The method described in any of paragraphs 25-27, wherein the fermentation product is ethanol.

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[37] . A method for producing a glucose amylase variant as described in any of paragraphs 1-19, the method comprising: culturing a host cell as described in paragraph 33 under conditions suitable for expression of the variant; and optionally recovering the variant.

[0255] The present invention is further described through the following examples.

[0256] Example Example 1: Glucoamylase activity can be measured in units of AGU.

[0257] Glucoamylase activity (AGU) Glucoamylase units (AGU) are defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute to produce glucose under standard conditions (37°C, pH 4.3, substrate: 100 mM maltose, buffer: 0.1 M acetate, reaction time: 6 minutes, as described in the following glucosylase incubation).

[0258] The analytical principle is described through three reaction steps: Step 1 is an enzymatic reaction: Glucoamylase (AMG) EC 3.2.1.3 (exo-α-1,4-glucan-glucosaminidase) hydrolyzes maltose to form α-D-glucose. The reaction is stopped with NaOH after incubation.

[0259] Steps 2 and 3 trigger the endpoint reaction.

[0260] In the reaction catalyzed by hexokinase, glucose is phosphorylated by ATP. The resulting glucose-6-phosphate is oxidized to 6-phosphogluconic acid by glucose-6-phosphate dehydrogenase. In this same reaction, an equimolar amount of NAD+ is reduced to NADH, resulting in an increase in absorbance at 340 nm. An automated analyzer system such as the Konelab 30 analyzer (Thermo Fisher Scientific) can be used.

[0261] Reagents used in the assay: A 1 M sodium acetate buffer stock solution was prepared by dissolving 44.4 g of sodium acetate trihydrate (Merck catalog number 61751805001730) and 37.5 ml of acetic acid (Fisher catalog number 11007) in Milli-Q water. The pH was adjusted to 4.3, and the final volume of the buffer was brought to 1000 ml. This buffer stock solution was stored at 4°C until use. A 100 mM working solution was prepared by adding 100 ml of the 1 M stock solution to 900 ml of Milli-Q water.

[0262] A fresh 0.1% substrate solution of 4-nitrophenyl-α-D-glucopyranoside (pNPG) was prepared by dissolving 100 mg of 4-nitrophenyl-α-D-glucopyranoside (Sigma catalog number N1377) in 100 ml of 100 mM sodium acetate buffer (pH 4.3).

[0263] A stock solution of 0.1 M borax (disodium tetraborate) termination solution was prepared by dissolving 38.1 g of borax (Fisher catalog number 27965) in 1000 ml of MilliQ water. This termination solution was stored at room temperature until use.

[0264] A fresh 1% maltose substrate solution was prepared by dissolving 1 g of maltose (Sigma catalog number M5885) in 100 ml of 100 mM sodium acetate buffer (pH 4.3).

[0265] A stock solution of 1000 µM acarbose was prepared by dissolving 64.6 mg of acarbose (Sigma catalog number A8980) in 100 ml of Milli-Q water. This stock solution was stored at 4°C until use. A 5.6 µM working solution was prepared by adding 336 µl of the 1000 µM stock solution to 59.66 ml of Milli-Q water.

[0266] Determination of specific activity (SA): Acarbose assays were used to determine the specific activity in the culture supernatant. This method uses a known concentration of acarbose that results in 50% inhibition of protein activity. The activity in the culture supernatant was normalized based on relative amylase glucosidase activity calculations (RAG) to determine the inhibition at the known concentration of acarbose. The resulting residual activity was then used to calculate the specific activity of amylase in the culture supernatant. Specific activity was calculated using the following equation.

[0267] Vsa = Vm x (1-Va / Vdw) Vm = A505 using a variant with a maltose substrate Va = A400 using a variant of acarbose Vdw = A400 variant without acarbose Specific activity of glucose amylase (SA) The specific activity of the purified protein was determined by AGU measurement using a Konelab instrument.

[0268] Determination of relative amylase (RAG) activity The RAG / ml of the culture supernatant for the variant and control was determined by relative amylase units (RAG).

[0269] The reaction mixture for RAG determination was prepared in a 384-well microtiter plate (Nunc, catalog number 262160). A 5 μl sample of unprocessed supernatant was added to 15 μl of 100 mM sodium acetate buffer (pH 4.3). 40 μl of 0.1% pNPG substrate was added to the plate and incubated at room temperature (25°C) for 15 min. The reaction was terminated by adding 30 μl of stop solution, and the absorbance was measured at 400 nm using an Infinite M1000 reader (TECAN, Switzerland).

[0270] Calculate the RAG / ml activity for each sample using the following equation: RAG / ml = ((SB) x F x AGs) / Ss-Bs S = Sample value B = Culture medium blank value Ss = Protein standard (0.6 AGU / ml) value Bs = Buffer Blank Value F = Dilution factor AGs = GU / ml of protein standard (0.6 AGU / ml) Normalization of culture supernatant Based on the initial RAG / ml of the samples, each uncooked supernatant sample was normalized to 0.6 RAG / ml with 100 mM sodium acetate buffer (pH 4.3), resulting in a final volume of 220 μl. The required volumes of uncooked supernatant and buffer for a single sample were calculated and normalized to 0.6 RAG / ml. For normalization, the calculated volume of 100 mM sodium acetate buffer (pH 4.3) was added to a 96-well microtiter plate (Nunc, catalog number 260836). Uncooked supernatant samples were added to the same 96-well microtiter plate and thoroughly mixed.

[0271] Maltose determination To assess the activity of the normalized sample against maltose, 10 μl of the normalized sample was mixed with 90 μl of 1% maltose substrate in a 96-well Abgene PCR plate (Thermo Scientific, catalog number AB0800) and incubated for 12 min at 45 °C in a programmable thermal cycler (T-ROBOT). Following incubation, 10 μl of the reaction mixture was mixed with 200 μl of Wako solution (LabAssay glucose, WAKO, catalog number 298-65701) in a 96-well microtiter plate (Nunc, catalog number 260836) and incubated at room temperature (25 °C) for 15 min. Absorbance was measured at 505 nm using an Infinite M1000 reader (TECAN, Switzerland).

[0272] Acarbose inhibition Acarbose inhibition was determined by normalizing the glucosylamylase (AMG) activity of the samples with and without acarbose (0.7 µM). 70 μl of the normalized sample was incubated with 10 μl of 5.6 μM acarbose in a 96-well microtiter plate (Nunc, catalog number 260836) at 25 °C for 10 min. AMG activity of the normalized samples incubated with and without acarbose was measured using pNPG as a substrate. 20 µl of the sample was transferred to a 384-well microtiter plate (Nunc, catalog number 262160) and mixed with 40 μl of 0.1% pNPG substrate. After incubation at 25 °C for 1 hour, the reaction was terminated by adding 30 μl of 0.1 M borax. The absorbance was read at 400 nm using an Infinite M1000 reader (TECAN, Switzerland).

[0273] These absorbance values ​​obtained from inhibition by maltose and acarbose are fitted into the above equation used to determine specific activity.

[0274] Heat transfer measurements used to determine thermal stability: Thermal change (Tm) was determined by measuring the thermal stability of proteins using a fluorescent protein-binding dye (SYPRO Orange; SIGMA S5692). SYPRO Orange nonspecifically binds to hydrophobic surfaces. When the protein is unfolded, the exposed hydrophobic surface binds to the dye, leading to increased fluorescence. Stability curves and the midpoint value (melting temperature, Tm) were obtained by gradually increasing the temperature to unfold the protein and measuring the fluorescence at each point.

[0275] Mix 5–10 µl of culture supernatant (GsAMG) with 17.5 µl of buffer (50 mM sodium acetate, pH 4.5) and 2.5 µl of 2.5X TAMRA dye (containing SYPRO Orange linked to the reporter gene). Maintain the total reaction volume at approximately 30 µL and prepare at room temperature. Finally, centrifuge the plate and cover it with an Applied Biosystems microAMP optical adhesion membrane (catalog number 4311971). Fluorescence-based heat transfer assays can be performed on an instrument that combines sample temperature control and dye fluorescence detection (7500 Rapid Real-Time PCR, Applied Biosystems). This instrument heats the sample in 50 mM sodium acetate buffer (pH 4.5) from 45°C to 78°C.

[0276] The reaction mixture was prepared in a 96-well Applied Biosystems microAMP rapid optical reaction plate (43669320). The melting temperature (Tm) of each variant, determined by TSA, was used as an indicator of thermal stability. An improvement factor (IF) for thermal stability was calculated relative to the average of the four wild-type variants on the plate (= Tm of individual variant / average Tm of wild-type). Tm values ​​were taken from the inflection point of the fluorescence spectrum (calculated using Protein Thermal Shift Software (Boltzmann method) (Applied Biosystems)). Variants with higher thermal stability were selected when compared to wild-type Tm.

[0277] Example 2: Determination of specific activity and chain-breaking temperature Variants according to the invention were produced, and their improvements were measured as increased specific activity determined by the acarbose assay described above and / or increased melting temperature determined by the heat transfer assay (TSA). The results are disclosed in the table below.

[0278] Table 1 discloses variants of increased thermal stability as measured by increasing melting temperature. The maximum observed increase is disclosed for each location.

[0279] Table 2. Reveals the increased specific activity of variants as measured by the increase in the improvement factor (IF), with the largest observed increase disclosed for each position.

[0280] Example 3. Further evaluation of the selected variant Minimum purification and specific activity determination Fifty-four candidates selected by acarbose titration underwent semi-purification and specific activity determination. The strains were cultured in shake flasks (Appendix 1), and 1 ml of culture supernatant was adsorbed onto α-CD agarose gels in 96-well plates with 100 mM acetate buffer (pH 4.0). The resin adsorbed with AMG was washed with 100 mM acetate buffer (pH 4.0) by centrifugation, and AMG was eluted with 10 mM β-CD in 100 mM acetate buffer (pH 4.0). Relative specific activity compared to Gs-WTAMG was determined by AMG activity assay (as described in Appendix 2 below) and by calculation of protein quantity determined by absorbance at 280 nm. Of the 54 samples tested, six samples (D4R, S5V, I13S, K15R, V18M, and V85G) showed improved specific activity.

[0281] The specific activity of the selected variant was determined. Strains expressing six candidate strains (D4R, S5V, I13S, K15R, V18M, and V85G) and wild-type Gs-AMG (glucosidase from *Entoloma viminalis*) were cultured in SF for sample preparation. The culture supernatant, filtered through a 0.2 μm sterile filter, was purified by affinity chromatography on an α-CD-coupled agarose gel. Details of the culture and purification procedures are described in Appendix 1. The specific activity of the purified samples was calculated using AMG activity determined by AGU assay (Appendix 2) and the amount of protein determined by absorbance at 280 nm. The improvement factor values ​​calculated according to the method in Appendix 2 cannot be directly compared with the IF values ​​calculated in Examples 1 and 2.

[0282] Characterization of combinatorial variants In conjunction with the variants of *Gymnospermum globulinum* glucoamylase previously described in WO 2016 / 062875 and WO 2014 / 177546, selected variants from Example 2 and some further confirmed variants (T43K, V18M, D4R, and S5V) from Example 3 above are introduced.

[0283] The tested substitution combinations were introduced into the Gs-AMG wt glucoamylase of SEQ ID NO: 3. Each expression plasmid was constructed by point mutation via PCR, and the constructed plasmids were used for transformation of the Aspergillus niger host strain. Transformants obtained by SF culture were purified and characterized by the culture supernatant to determine specific activity (SA) and denaturation temperature (Td) by thermal change assay (Appendix 3).

[0284] The characterization results are summarized in the table below.

[0285] Appendix 1 SF cultivation The strains were inoculated onto COVE-N-gly plates and incubated at 30°C for one week. Then, 1 cm... 2 The mycelia were inoculated into 100 ml of MSS in a 500 ml shake flask and cultured at 30°C and 200 rpm for 3 days. Then, 10 ml of seed culture was inoculated into 100 ml of MU-1 in a 500 ml shake flask and cultured at 30°C and 200 rpm for 6 days.

[0286] Acarbose affinity chromatography Sample preparation If necessary, adjust the pH of the sample to pH 4-5 by adding 2 M sodium acetate buffer to 50 mM.

[0287] Before loading the column, filter the sample using a 0.22 μm PES membrane. Keep the sample at 4°C before loading.

[0288] Chromatography Chromatographic conditions System: Akta explorer 10S equipped with an air sensor Column: α-CD-coupled agarose gel, 35 ml column volume (CV), packed in a glass column (GE Healthcare, 26 mm inner diameter (id) x variable height, with protective shield). Equilibration buffer (Buffer A): 50 mM NaOAc, 150 mM NaCl, pH 4.5 Elution buffer (Buffer B): 50 mM NaOAc, 150 mM NaCl, 10 mM β-cyclodextrin, pH 4.5 # The buffer solution is filtered through a 0.22 μm PES membrane and degassed by a vacuum aspirator with ultrasonic application before use.

[0289] Flow rate: 5 ml / min fraction size: 10 ml Sample volume: 5 ml – 1000 ml program 1. Equilibrate with buffer A, 3CV 2. Load the sample via a sampling pump, 5 ml - 1000 ml 3. Wash the column with buffer A, 3CV 4. Elute with buffer B, 3CV (For multiple samples) 5. Regenerate the column with 0.1M NaOH, 3CV Merging grades Preferably, only one AMG peak is observed during the elution step. Fractions are collected based on the A280 peak.

[0290] Buffer swap Using MWCO:12000 dialysis tubes, the combined fractions were buffer-exchanged to 20 mM sodium acetate at pH 4.5 by overnight dialyzing with 10 L of buffer at 4°C, and the sample was concentrated to the appropriate volume using an ultrafiltration membrane (e.g., Vivacel 250 equipped with a MWCO 5000 membrane (10000 is not recommended) or Amicon Ultra YM-15 MWCO 12000).

[0291] Appendix 2 The analytical principle is described through three reaction steps: Step 1 is an enzymatic reaction: Amyl glucosidase (AMG) EC 3.2.1.3 (exo-α-1,4-glucan-glucose hydrolase) hydrolyzes maltose to form α-D-glucose. The reaction is stopped with NaOH after incubation.

[0292] Steps 2 and 3 lead to an endpoint reaction: In a reaction catalyzed by hexokinase, glucose is phosphorylated by ATP. The resulting glucose-6-phosphate is oxidized to 6-phosphogluconic acid by glucose-6-phosphate dehydrogenase. In this same reaction, an equimolar amount of NAD+ is reduced to NADH, resulting in an increase in absorbance at 340 nm.

[0293] Measurement Procedure 100 μl of substrate (100 mM maltose in 100 mM acetate buffer at pH 4.3) was incubated at 37°C for 8 minutes. 20 μl of sample was added to the pre-incubated substrate, and the mixture was incubated for 6 minutes. The reaction was terminated by adding 20 μl of NaOH. 110 μl of GHK reagent (Konelab from Thermo Fisher Scientific) was added. TM Add System glucose reagent (catalog number 981304) to the reaction mixture and incubate for 7 minutes. Then, determine the absorbance at 340 nm.

[0294] Appendix 3 The denaturation temperature (Td) was measured by heat transfer assay (TSA). The purified enzyme was diluted to 0.25 mg / ml with deionized water, and the SYPRO Orange fluorescent dye (Invitrogen) was diluted 1666-fold with deionized water. The diluted enzyme and dye were mixed 1:1, and 30 μl of the mixture was transferred to the wells of a 96-well white PCR plate. The temperature was linearly increased from 37 °C to 96 °C, and the fluorescence changes in each well were monitored using a real-time PCR device (LightCycler 480, Roche Diagnostics). The operating parameters are shown in the table below. The obtained curves (signal versus temperature) were normalized so that the local minimum and local maximum of the signal were 0 and 1, respectively, and the temperature at which a normalized signal of 0.5 was given was defined as the denaturation temperature (Td).

Claims

1. A glucose amylase variant comprising substitutions at one or more sites selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, I13, K15, V18, L19, N25, S27, K28, S30, V36, V3 7. T43, D45, S57, V59, F60, I71, S73, T74, L77, D82, D83, V85, T86, E88, L9 1. S95, P97, T103, D114, S134, L137, T139, N142, L145, S146, N147, N149 , Y152, V153, T154, S155, N156, L157, W158, P159, I160, Q162, V169, S170 , S175, T176, Y177, D184, S186, R199, A202, A203, T206, Q210, T211, S21 2. Q213, V214, S215, Y217, T218, T219, Q220, A221, D222, N223, L224, F22 7. Y231, P234, S235, Y238, T240, T243, G244, G245, G246, R247, S248, A2 52. T254, L255, Y262, S265, G267, A270, A271, K279, S282, L284, V294, Y2 95, S296, I297, N298, S299, G300, A302, S303, N304, T309, E314, S316, Q 318, G319, T326, V330, N339, E342, S343, E348, S351, T352, Q359, S362, G 363, V364, T365, A366, S371, S372, T378, S381, I383, N385, F386, A392, N394, K396, Y408, K410, D412, S414, S417, V419, A426, S427, E433, A434, N436, N437, T438, Q439, G442, A446, L448, V450, N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N495, S501, A502, T506, I509, T510, N512, S516, A518, I519, N527, N528, A530, E534, D536, P537, N538, N539, I541, A545, S546, G547, S548, N552, and T554, wherein the positions correspond to the amino acid positions in the amino acid sequence shown in SEQ ID NO: 3;Furthermore, the substitutions at one or more positions provide an increased glucosylamylase variant with a melting temperature of at least 0.5 degrees Celsius as measured by TSA, and further, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO:

3.

2. The glucosylamylase variant of claim 1, wherein the variant comprises substitutions at one or more sites selected from the group consisting of: Q1K、Q1R、S2E、S2K、S2L、S2P、S2R、V3L、V3G、V3R、D4R、D4S、D4G、D4A、D4W、S5L、S5V、S5G、S5C、S5R、S8Q、S8H、S8A、S8Y、S9C、S9Q、S9M、S9W、S9D、S9G、I13V、I13R、I13S、I13L、I13E、K15G、K15R、V18M、V18Q、L19G、L19F、N25S、N25A、S27A、S27L、S27G、S27V、S27C、K28C、K28R、S30Q、S30A、S30K、S30T、S30L、V36K、V36G、V36W、V36A、V36I、V37R、V37K、V37G、V37C、V37M、V37S、V37T、V37D、T43K、D45L、D45P、S57P、S57L、S57G、S57F、S57R、S57T、S57A、V59T、V59S、V59E、F60S、I71M、I71S、I71T、I71V、S73H、S73R、S73N、S73V、S73G、T74V、L77S、L77P、L77R、D82N、D82R、D82V、D82G、D83L、D83C、D83W、V85Q、V85G、V85P、T86R、T86V、E88Q、E88R、E88G、L91S、L91P、L91G、S95A、S95T、S95V、P97T、P97I、P97R、T103Y、T103A、T103G、D114G、D114N、D114M、D114R、D114C、S134P、S134A、S134V、S134W、S134D、S134H、S134L、S134G、L137W、L137S、L137A、L137V、L137G、L137D、L137R、L137P、T139D、T139P、T139V、N142Y、N142H、N142C、L145C、L145D、L145G、L145V、L145S、S146W、S146L、S146R、S146G、S146P、N147Q、N147V、N147L、N147K、L147D、L147Y、L147H、L147S、N149H、N149T、N149R、N149K、N149S、Y152S、Y152A、Y152R、Y152L、Y152K、Y152E、Y152P、Y152V、Y152I、Y152C、Y152W、V153E、V153S、V153G、V153W、V153Y、T154N、T154R、T154K、T154P、T154V、S155C、S155P、S155R、S155G、S155A、N156I、N156A、N156V、N156R、N156T、N156K、L157P、L157R、L157A、L157G、L157W、W158T、W158A、W158M、W158V、W158R、W158P、P159S、P159G、P159L、P159V、P159A、P159R、P159Q、P159E、I160A、I160G、I160N、I160T、I160R、I160V、Q162L、Q162V、Q162H、Q162P、Q162R、V169A、V169L、V169W、V169S、V169D、V169R、V169E、S170A、S170P、S170R、S170M、S175L、S175C、S175W、T176R、T176L、T176N、T176A、T176S、T176I、Y177H、D184P、D184W、D184S、D184Y、D184G、S186A、S186R、S186W、R199F、R199E、R199L、R199C、R199K、A202R、A202W、A202E、A202S、A202V、A203M、A203W、A203P、A203L、T206C、T206P、T206G、T206A、T206R、Q210C、Q210G、Q210S、Q210R、Q210L、Q210P、Q210V、T211R、T211A、T211H、T211K、T211Q、T211G、T211W、T211E、T211I、T211V、T211P、T211L、T211D、S212D、S212E、S212L、S212P、S212T、Q213W、Q213V、Q213D、Q213A、Q213T、Q213R、Q213G、Q213S、V214G、V214R、V214W、V214A、V214I、S215R、S215G、S215L、S215Y、S215P、S215E、S215W、Y217G、Y217C、Y217A、Y217S、Y217T、Y217F、T218H、T218C、T218A、T218M、T218Q、T218G、T219R、T219D、T219S、T219G、T219C、Q220R、Q220V、Q220D、Q220S、Q220L、A221V、A221T、A221L、A221P、A221R、A221E、D222V、D222W、D222T、D222G、D222L、D222R、D222N、D222F、D222M、N223A、N223S、N223R、N223F、N223P、N223G、N223L、L224G、L224D、L224K、L224V、L224R、F227G、F227W、Y231S、Y231T、Y231R、Y231L、Y231A、Y231V、Y231N、P234D、P234L、P234S、P234V、S235C、S235R、S235N、S235G、S235W、Y238R、Y238A、Y238Q、Y238C、Y238E、T240C、T240I、T240L、T240S、T243V、T243S、T243L、T243R、G244R、G244C、G244P、G244D、G244W、G245R、G245S、G245V、G245W、G245M、G246L、G246E、G246S、G246R、G246K、G246W、G246D、R247E、S248Y、S248P、S248V、S248L、S248F、S248A、S248E、S248W、S248K、S248T、A252E、A252T、A252Y、A252V、A252L、T254D、T254W、T254V、T254G、T254A、L255R、L255Q、L255P、L255G、Y262C、Y262Q、Y262S、Y262G、Y262V、Y262A、Y262W、S265C、S265P、S265G、S265L、G267W、G267C、A270L、A270M、A271W、A271Y、A271L、K279R、K279W、K279E、K279P、K279G、K279F、S282W、S282T、S282K、S282R、L284N、L284Q、L284T、L284S、L284R、L284G、L284V、V294G、V294W、V294E、V294S、Y295V、Y295R、S296F、S296L、S296W、S296K、I297S、I297P、I297K、I297F、I297R、I297W、N298W、N298G、N298C、N298V、N298L、N298A、S299P、S299C、S299M、S299L、S299T、G300S、G300A、G300P、G300L、G300W、A302G、A302L、A302C、A302R、A302V、S303P、S303V、S303C, S303A, S303R, N304T, N304R, N304Q, N304L, N304V, T309G, T309I, T309R, T309M, E314Y, E314T, E314V, E314G, E314S, E314L, E314A, S316T, S316 L, S316G, S316F, S316R, S316P, S316V, S316Q, Q318L, Q318R, G319R, G319Q, G319P, G319A, T326V, T326G, T326W, T326N, T326A, V330S, V330L, V330P, V33 0R、V330A、V330G、N339P、N339A、N339T、E342M、E342W、E342N、E342L、E342R 、S343R、S343C、E348W、E348F、E348P、E348V、E348G、E348M、S351P、S351C、S 351G, S351R, S351L, S351W, T352P, T352L, T352G, T352Q, T352Y, Q359K, Q359P, Q359R, Q359S, Q359A, S362P, S262R, S262G, S262M, G363R, G363T, G363P V364A, V364C, V364E, V364S, V364G, V364L, T365S, T365G, T365W, T365L, T365H, A366D, A366T, A366P, A366R, A366H, S371A, S371G, S372A, S372E, S372 C, S372L, S372R, T378G, T378L, T378D, T378H, T378A, T378P, S381K, I383A, I383G, I383C, I383L, I383T, I383M, N385R, N385W, N385S, N385G, N385D, F38 6S、F386W、F386Q、F386V、F386I、F386G、F386C、F386A、F386T、F386L、A392V 、A392L、A392E、A392G、N394D、N394R、N394Y、N394W、N394E、K396I、K396W、K 396P、K396Y、K396F、Y408V、Y408E、Y408P、Y408S、Y408K、Y408L、K410S、K41 0R、D412M、D412S、D412N、D412W、D412L、D412R、S414C、S414R、S414G、S414V、S414W、S414H、S417Y、V419S、V419G、V419C、V419A、V419K、V419R、V419T、A426M、A426N、A426K、A426R、S427G、S427A、S427P、S427N、S427D、S427L、E433C、A434Q、A434G、N436S、N436P、N436D、N437K、N437R、N437T、N437P、T438E、T438G、Q439W、Q439S、Q439G、Q439C、Q439R、Q439Y、G442V、G442D、G442C、G442A、G442L、G442W、G442E、G442M、G442R、A446G、A446D、A446R、A446E、A446I、L448G、L448P、L448E、V450P、V450S、V450C、V450E、V450L、V450N、N470H、N470D、N470K、N470V、N470L、E472I、V474W、V474C、V474A、V474L、V474G、W475P、W475A、W475R、N478L、N478I、N478P、N478R、N478W、N478S、N478G、N478K、N478A、S484G、S484Y、S484P、S484A、S484N、V485A、V485W、V485K、V485G、V485R、D486I、D486K、D486Y、D486S、D486A、D486W、D486L、A487S、A487V、A487L、A487G、A487C、A487K、S492L、S492R、S492T、S492W、S492P、S492C、A493V、A493R、A493D、A493W、D494N、D494R、D494G、D494L、D494E、D494Q、N495L、N495W、N495G、N495R、N495C、S501R、S501L、S501M、S501K、S501W、A502C、A502Q、A502W、A502G、A502V、T506A、T506P、T506V、I509E、I509D、I509S、I509F、I509W、I509R、T510F、T510E、T510R、T510P、T510V、T510A、T510L、N512Q、N512K、N512H、N512R、N512V、S516R、S516W、S516P、S516K、S516Y、S516C、A518D、A518G, A518Y, A518V, A518R, A518L, A518T, I519W, I519L, I519R, I519F, I519K, N 527T, N527K, N527P, N527L, N528D, N528G, N528K, N528V, N528E, N528L, A530R, A53 0C, A530G, A530V, A530S, A530T, E534W, E534Q, E534C, E534V, E534G, E534R, E534 F. E534K, D536G, D536R, D536W, D536H, D536K, D536N, D536M, D536C, D536V, P537D, P537M, P537W, P537G, P537E, N538D, N538S, N538W, N538Y, N538A, N539M, N539R, N 539P, N539A, I541A, I541T, I541V, I541G, I541N, A545R, A545T, A545V, A545L, S54 The substitutions at one or more positions provide a glucosylamylase variant with an increased melting temperature of at least 0.5 degrees Celsius as measured by TSA, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence having SEQ ID NO:

3.

3. A glucose amylase variant comprising substitutions at one or more sites selected from the group consisting of: Q1, S2, V3, D4, S5, S8, S9, G11, I13, K15, A16, V18, L19, N25, S27, S30, A32, A34, V36, V37, S44, S57, V59, F60, Y67, T68, I71, D72, S7 3. T74, S75, S76, L77, R78, D82, D83, F84, V85, T86, N90, L91, Q93, S95, L101, T102, T103, S134, L137, T139, N142, L145, S146, N147, Y152, V153, T154, S155, L157, W158, P159, I160, Q162, N163, S170, S175, T176, Y177, S186, R199, A202, A203, T206, Q210, T211, S21 2. Q213, S215, Q220, A221, D222, N223, L224, F227, P234, S235, Y238, T240, T243, G244, G245, G246, S248, A252, T254, L255, A270, A 271, K279, S282, L284, Y295, S296, I297, N298, S299, G300, A302, S303, N304, S316, G319, T326, V330, N339, E342, S343, Q344, E348 , S351, Q359, S362, G363, T365, A366, S371, S372, T378, S381, I383, F386, A392, N394, K396, N401, K410, D412, S414, S417, V419, D4 20, E433, N437, T438, Q439, F440, G442, A446, N470, E472, V474, W475, N478, S484, V485, D486, A487, S492, A493, D494, N495, S501, A502, I509, T510, N512, S516, A518, I519, N527, A530, E534, P537, N538, N539, I541, A545, S546, G547, N552, and T554, wherein the positions correspond to amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a value of at least 1 as measured as an improvement factor IF.A glucosylamylase variant with increased specific activity of 1, and further wherein said variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO:

3.

4. The glucosylamylase variant of claim 3, wherein the variant comprises substitutions at one or more sites selected from the group consisting of: Q1R、Q1L、Q1T、Q1G、Q1P、Q1K、Q1M、Q1F、Q1S、Q1A、Q1W、S2V、S2Q、S2E、S2D、S2P、S2A、S2T、S2L、S2R、S2K、S2W、S2G、V3G、V3L、V3I、V3A、V3E、D4R、D4C、D4S、D4G、D4N、D4V、D4W、D4F、D4A、S5V、S5R、S5P、S5L、S5G、S5C、S5N、S5Q、S5T、S8A、S8W、S8R、S8L、S8Y、S8G、S8M、S8H、S8P、S8Q、S8V、S8C、S8E、S8K、S8T、S9D、S9Q、S9R、S9G、S9A、S9N、S9E、S9K、S9L、S9T、S9M、G11D、I13L、I13A、I13Q、I13S、I13D、I13R、I13M、I13V、I13G、I13Y、I13E、K15V、K15R、K15I、K15M、K15A、K15F、K15L、K15S、K15E、K15W、K15G、K15D、A16L、A16V、A16G、A16E、A16S、A16T、A16K、A16G、V18A、V18R、V18M、V18T、V18L、V18Q、V18I、L19S、L19A、L19K、L19V、L19C、L19H、L19W、L19F、L19R、N25W、N25Y、N25D、N25F、N25G、N25R、N25V、N25L、N25A、N25S、N25E、N25C、N25Q、S27A、S27W、S27H、S27V、S27T、S27C、S27G、S27E、S27L、S27F、S30A、S30P、S30K、S30R、S30Q、S30Y、S30E、S30D、S30T、S30V、A32D、A32E、A32S、A32V、A32R、A32G、A32M、A32T、A32C、A32K、A32W、A34W、A34R、A34L、A34Q、A34G、A34C、A34F、A34V、A34E、A34T、A34I、A34P、V36I、V36R、V36A、V36G、V36L、V37C、V37G、V37R、V37A、V37M、S44R、S44W、S44L、S44T、S44C、S44A、S44V、S44P、S44E、S57G、S57T、S57H、S57P、S57A、V59T、V59E、V59Q、V59L、V59R、、F60S、F60V、F60A、F60I、Y67C、Y67N、Y67A、Y67G、Y67T、Y67V、Y67D、Y67H、Y67R、Y67F、Y67L、Y67P、Y67S、Y67M、T68K、T68C、T68A、T68P、T68R、T68Q、I71T、I71M、I71V、I71S、I71N、I71F、I71D、I71P、I71R、I71L、I71K、D72L、D72G、D72N、D72R、D72K、D72E、D72W、D72A、D72C、D72Y、D72S、D72Q、D72T、S73H、S73G、S73N、S73C、S73R、S73V、S73L、S73I、S73W、S73P、T74S、T74E、T74P、T74N、T74F、T74P、T74M、T74R、T74C、S75G、S75N、S75P、S75E、S75C、S75R、S75L、S75K、S75I、S75T、S76H、S76P、S76Q、S76E、L77S、L77Y、L77E、L77P、R78W、R78G、R78K、R78Q、R78T、R78A、R78C、R78M、R78E、D82V、D82G、D82R、D82N、D82E、D82C、D83L、D83C、D83W、D83A、D83R、D83G、D83V、D83S、D83E、F84Y、F84L、F84S、F84T、F84P、F84E、F84V、F84A、F84W、F84K、F84M、F84R、V85G、V85W、V85P、V85Q、V85E、V85H、V85R、V85T、T86C、T86R、T86G、T86W、T86D、T86V、T86S、T86A、N90G、N90E、N90T、N90P、N90C、L91H、L91P、L91F、L91V、L91R、Q93L、Q93M、Q93C、Q93H、Q93G、Q93R、Q93W、Q93D、Q93A、Q93N、Q93K、S95V、S95R、S95D、S95Y、S95G、S95Q、S95A、S95K、L101M、L101V、L101R、L101P、L101F、L101H、L101A、L101G、L101N、L101K、L101C、T102N、T102S、T102C、T102R、T102A、T102I、T102M、T102W、T102E、T102P、T102F、T103A、T103S、T103G、T103D、T103I、T103E、T103V、T103N、S134V、S134I、S134M、S134P、S134L、S134A、S134C、L137S、L137D、L137W、L137G、L137R、L137A、L137I、L137T、T139A、T139N、T139S、T139G、T139D、T139H、T139R、N142K、N142E、N142Q、N142R、N142G、N142H、N142W、N142A、L145S、L145W、L145N、L145C、L145V、L145R、L145D、S146V、S146G、S146L、S146T、S146A、S146C、S146P、S146F、S146R、S146W、N147K、N147E、N147S、N147F、N147T、N147I、N147D、N147P、N147Y、N147H、N147L、Y152V、Y152E、Y152L、Y152I、Y152A、Y152M、Y152R、Y152F、Y152G、V153R、V153Y、V153C、T154R、T154G、T154L、T154S、T154A、T154M、T154P、S155R、S155G、S155L、S155A、S155H、S155W、S155C、S155I、S155P、S155M、S155N、S155T、L157P、L157Q、L157V、L157M、L157R、W158R、W158E、W158C、W158K、W158L、W158G、P159S、P159R、P159V、P159Q、P159T、P159D、P159A、P159L、P159G、I160T、I160A、I160V、I160D、I160G、I160S、I160L、I160Y、I160N、I160F、Q162L、Q162K、Q162R、Q162S、Q162H、Q162P、Q162I、Q162V、N163D、N163G、N163R、N163T、N163I、N163Q、N163Y、N163K、N163H、N163W、N163A、N163S、S170A、S175W、S175R、S175T、S175C、T176S、T176R、T176L、T176A、T176W、T176I、Y177S、Y177T、Y177D、Y177V、S186V、S186R、S186E、S186L、S186D、S186C、S186A、S186Q、R199K、R199V、R199A、R199M、R199N、R199W、R199T、R199E、A202S、A202T、A202Q、A202L、A202E、A202P、A202V、A202F、A202W、A202G、A203Q、A203K、A203W、A203R、A203V、A203L、A203M、A203T、A203E、A203G、A203S、A203P、T206I、T206S、T206W、T206V、T206A、T206P、T206G、T206R、Q210D、Q210R、Q210G、Q210A、Q210L、Q210H、Q210P、Q210V、Q210I、Q210C、T211P、T211R、T211S、T211D、T211Q、T211H、T211A、T211L、T211G、T211W、S212V、S212K、S212D、S212T、S212H、S212L、S212P、S212E、S212C、S212A、S212M、Q213Y、Q213D、Q213R、Q213N、Q213S、Q213W、Q213K、Q213L、Q213C、Q213P、S215L、S215T、S215Q、S215R、S215V、S215G、S215N、S215C、Q220L、Q220P、Q220K、Q220R、Q220H、Q220E、A221V、A221T、A221E、A221G、A221P、D222E、D222M、D222A、D222G、D222N、D222V、D222H、N223K、N223R、L224V、F227A、F227V、F227L、F227S、F227Y、F227E、F227G、P234A、P234L、P234Q、P234S、S235C、S235R、S235W、S235G、S235K、Y238C、Y238L、Y238E、Y238W、Y238A、Y238S、Y238G、T240L、T240C、T240G、T240W、T240V、T240R、T240S、T240A、T240E、T243S、T243Q、T243M、T243G、T243L、T243V、T243E、T243P、T243R、T243W、G244W、G244D、G244Y、G244A、G244S、G244R、G245M、G245N、G245S、G245T、G245V、G245D、G245I、G246V、G246W、G246M、G246E、G246N、G246Q、G246S、G246D、G246R、S248E、S248L、S248C、S248G、S248P, S248F, S248T, A252S, A252T, A252V, A252P, A252G, T254A, T254S, T254G, T254P, L255V, L255A, L255P, L255I, L255C, A270W, A270T, A270E, A270 C, A270M, A270S, A270L, A270G, A270R, A270Y, A270V, A271R, A271P, A271L, A271W, A271G, A271T, K279V, K279W, K279A, K279L, K279R, K279E, K279Y, K27 9P, K279G, K279S, S282G, S282T, S282L, S282V, S282F, S282R, S282A, S282I, S282W, L284V, L284G, L284S, L284M, L284T, Y295K, Y295H, Y295Q, Y295W, Y 295M, Y295F, Y295C, Y295E, Y295V, S296A, S296T, S296K, S296N, S296Y, S296F, S296Q, S296P, S296L, S296D, I297L, I297V, I297H, I297R, I297W, I297K I297T, I297F, I297G, I297Q, N298M, N298D, N298S, N298R, N298K, N298A, N298V, N298E, N298G, N298L, S299L, S299G, S299V, S299A, S299R, S299Q, S299 M、S299I、S299P、S299T、G300A、G300N、G300D、G300R、G300L、G300F、G300C、 G300P、G300W、G300T、G300S、A302L、A302R、A302P、A302V、A302K、A302M、A30 2Y, A302S, A302T, A302G, S303P, S303K, S303R, S303C, S303A, S303F, S303W, S303L, S303Q, N304V, N304G, N304P, N304W, N304F, N304E, N304T, N304D, N 304R, N304S, N304A, N304I, N304M, N304K, S316T, S316C, S316A, S316R, S316P, S316H, S316K, S316F, S316G, S316Q, S316N, S316M, S316L, S316V, G319TG319R, G319W, G319S, G319Q, G319A, G319D, T326S, T326G, T326A, T326C, T326Y, T326P, T326I, T326E, T326Q, V330M, V330G, V330I, V330D, V330P, V330 L, V330Y, V330S, V330A, N339T, N339R, N339S, N339A, N339Q, N339P, E342L, E342K, E342T, E342M, E342R, E342V, E342H, E342G, E342Q, E342S, E342F, E34 2A, E342W, S343A, S343W, S343G, S343P, S343Q, S343T, S343E, S343R, S343L, Q344L, Q344V, Q344T, Q344D, Q344A, Q344H, Q344K, Q344R, Q344P, Q344E, E 348C、E348G、E348V、E348M、E348N、E348A、E348I、E348D、E348L、E348K、E34 8R、S351Y、S351G、S351R、S351C、S351N、S351L、S351K、S351V、S351F、S351T、 S351A, S351P, S351W, Q359A, Q359V, Q359T, Q359R, Q359G, Q359L, Q359K, Q359S, Q359P, Q359W, S362V, S362P, S362R, S362G, S362H, S362E, S362M, S362 D, S362Y, S362C, S362F, S362A, S362Q, G363C, G363H, G363D, G363W, G363R, G363Q, G363S, G363A, G363T, G363P, T365R, T365W, T365G, T365L, T365C, T36 5Q, T365I, T365V, T365Y, T365S, T365E, A366R, A366L, A366I, A366Q, A366P, A366T, A366S, A366E, A366G, A366D, A366W, A366H, S371V, S371R, S371A, S 371T, S371G, S371C, S371E, S371P, S372P, S372E, S372R, S372A, S372Q, S372N, S372G, S372R, S372L, S372V, S372M, S372C, S372W, T378P, T378A, T378KT378W, T378M, T378Q, T378G, T378V, T378E, T378S, T378R, T378L, T378C, T378I, T378D, S381E, S381Y, S381D, S381N, S381R, S381G, S381V, S381A, S381 T、S381P、S381W、S381Q、S381C、S381I、I383F、I383N、I383G、I383C、I383E、 I383L、I383M、I383V、I383A、I383T、I383R、I383S、F386L、F386Y、F386R、F38 6S, F386G, F386M, F386C, F386W, F386A, A392V, A392R, A392T, A392S, A392E, A392L, A392G, A392P, A392F, A392M, A392I, A392Q, N394A, N394S, N394T, N 394R, N394H, N394G, N394C, N394E, N394W, N394P, N394L, N394V, N394F, N394Q, N394K, K396S, K396P, K396M, K396F, K396Q, K396E, K396D, K396W, K396L K396A、K396I、K396R、K396G、K396C、K396V、N401Q、N401V、N401F、N401S、N4 01T、N401G、N401R、N401C、N401A、N401D、N401K、N401E、N401Y、N401W、N401 P、N401L、K410S、K410T、K410L、K410D、K410M、K410V、K410P、K410N、K410C、 K410G、K410E、K410W、K410R、D412R、D412Q、D412S、D412P、D412E、D412N、D41 2G、D412V、D412L、D412W、D412A、D412K、D412M、D412T、S414P、S414A、S414W 、S414G、S414L、S414R、S414E、S414N、S414T、S414Q、S417R、S417G、S417K、S 417Y, S417A, S417N, V419D, V419E, V419A, V419G, V419M, V419L, V419I, D420V, D420A, E433W, E433P, E433M, E433Y, E433S, E433C, E433G, E433A, E433RE433Q、E433K、N437V、N437E、N437D、N437M、N437T、N437A、N437S、N437W、N437L、N437P、N437Y、N437G、N437Q、N437K、N437R、T438R、T438A、T438K、T438W、Q439A、Q439R、Q439G、Q439W、Q439P、Q439C、Q439M、Q439Y、Q439D、F440T、F440L、F440W、F440E、F440S、G442V、G442L、G442D、G442A、G442C、G442S、G442F、G442M、G442I、G442Y、G442W、A446L、A446R、A446F、A446G、A446S、A446M、A446Q、A446W、A446V、A446P、A446D、N470W、N470G、N470L、N470S、N470P、N470Y、N470A、N470E、N470D、N470H、N470K、N470T、N470M、E472W、E472S、E472L、E472G、E472R、E472P、E472V、E472T、E472K、V474R、V474F、V474Y、V474I、V474M、V474W、V474E、V474Q、V474L、V474G、V474A、V474K、V474T、V474H、W475P、W475S、W475L、W475C、W475Q、W475G、W475R、W475T、N478V、N478A、N478S、N478T、N478R、N478K、N478G、N478L、N478M、N478I、N478D、N478W、N478E、S484Q、S484T、S484E、S484F、S484A、S484G、S484D、S484L、S484W、S484V、S484R、S484Y、S484P、S484M、V485L、V485T、V485A、V485S、V485R、V485G、V485I、V485E、V485D、V485F、V485K、D486I、D486G、D486R、D486E、D486S、D486A、D486T、D486K、D486F、D486M、D486Q、D486C、D486L、D486Y、D486P、A487M、A487E、A487V、A487S、A487C、A487G、S492L、S492P、S492V、S492R、S492Y、S492M、S492H、S492T、S492K、S492W、A493G、A493S、A493Y、A493V、A493T、A493E、A493Q、A493R、D494A、D494S、D494E、D494Q、D494Y、D494G、D494R、D494T、D494W、D494N、D494H、D494L、D494M、D494V、D494P、N495S、N495L、N495F、N495C、N495W、N495R、N495G、S501P、S501T、S501L、S501G、S501M、S501R、S501K、S501V、S501E、S501A、S501C、A502W、A502V、A502S、A502G、A502D、A502E、A502T、A502M、A502Y、A502H、I509G、I509R、I509W、I509A、I509V、I509L、I509S、I509P、I509T、I509E、I509H、I509N、T510R、T510I、T510A、T510H、T510S、T510Y、T510V、T510L、T510K、T510E、T510P、T510F、T510M、N512S、N512Q、N512L、N512G、N512W、N512I、N512M、N512Y、N512K、N512V、N512H、N512F、N512T、N512R、N512D、S516Y、S516R、S516P、S516T、S516G、S516V、S516N、S516L、S516F、S516M、S516A、S516W、S516C、S516K、A518G、A518P、A518W、A518V、A518R、A518L、A518M、A518F、A518Y、A518S、I519L、I519C、I519G、I519W、I519S、I519Y、I519N、I519A、I519V、I519Q、I519T、I519H、I519M、N527S、N527L、N527V、N527G、N527W、N527H、N527R、N527K、A530R、A530C、A530S、A530G、A530F、A530Y、A530W、A530T、A530V、E534M、E534A、E534V、E534W、E534C、E534R、E534T、E534L、E534G、E534F、E534S、E534Q、E534K、P537R、P537T、P537H、P537M、P537G, P537A, P537S, P537E, P537Y, P537L, P537V, N538G, N538V, N538R, N538A, N538W, N538D, N538M, N538S, N 538I, N538Y, N539L, N539S, N539A, N539I, N539V, I541A, I541G, I541T, I541W, I541K, I541V, I541N, I541F, A54 5L, A545W, A545V, A545S, A545G, A545R, A545T, A545P, S546E, S546C, S546G, S546N, S546V, G547S, G547V, G547L, G547D, G547R, G547C, G547M, N552V, N552E, N552D, N552G and T554A, T554G, T554E, T554D, T554C, wherein the position corresponds to SEQ The amino acid positions in the amino acid sequence shown in SEQ ID NO: 3; and wherein the substitutions at one or more positions provide a glucosylamylase variant with an increased specific activity of at least 1.1 as measured by the improvement factor IF, and further wherein the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity with the amino acid sequence having SEQ ID NO:

3.

5. A variant according to any one of claims 1-4, wherein the variant comprises a substitution corresponding to position 43, specifically T43K and wherein the increase in melting temperature compared to the melting temperature of the glucosylamylase of SEQ ID NO: 3 is at least 2°C, for example at least 3°C.

6. A variant of any one of claims 1-4, wherein the variant comprises a substitution corresponding to a position selected from the group consisting of: Positions 4, 5, 13, 15, 18, 85, specifically, are substitutions selected from the following: D4R, S5V, I13S, K15R, V18M, V85G, wherein said variants have improved specific activity compared to the glucosylamylase of SEQ ID NO:

3.

7. The variant of any one of claims 1-6, wherein the variant further comprises substitutions corresponding to S95P and A121P.

8. A variant of any one of claims 1-7, wherein the variant further comprises a substitution corresponding to S95P + A121P + Y295W or S95P + A121P + Y295W + Q318Y.

9. A variant as claimed in any of the preceding claims, wherein the variant comprises at least one of the following substitutions or combinations of substitutions: T43K; D4R; S5V; I13S; K15R; V18M; V85G; S95P + A121P + Y295W + T43K; T43K + S95P + A121P + Y295W + Q318Y; V18M + T43K + S95P + A121P + Y295W + Q318Y; D4R + T43K + S95P + A121P + Y295W + Q318Y; S5V + T43K + S95P + A121P + Y295W + Q318Y; I13S + T43K + S95P + A121P + Y295W + Q318Y; V18M + T43K + S95P + A121P + Y295W; The variant is said to have glucosylamylase activity and the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 3, and the variant has increased specific activity and / or an increased melting temperature of at least 2°C, specifically at least 3°C, as measured by TSA, compared with the glucosylamylase of SEQ ID NO:

3.

10. The variant of any one of claims 1-9, wherein the number of changes is 1-20, such as 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes.

11. A composition comprising a glucose amylase variant as described in any one of claims 1-10.

12. Use of a polypeptide as described in any one of claims 1-10 for the production of syrups and / or fermentation products.

13. A method for producing fermentation products from starch-containing materials, the method comprising the following steps: (a) liquefying a starch-containing material in the presence of α-amylase; (b) saccharifying the liquefied material; and (c) fermenting the material with a fermenting organism, wherein step (b) is performed using at least one variant of glucosylamylase as described in any one of claims 1-10.

14. The method of claim 13, wherein steps (b) and (c) are performed simultaneously.

15. A method for producing fermentation products from starch-containing materials, the method comprising the following steps: (a) Saccharifying the starch-containing material at a temperature below the initial gelatinization temperature of the starch-containing material; and (b) Fermentation using fermentation organisms. Step (a) is performed using at least one variant of glucosyl amylase according to any one of claims 1-9.

16. A method for producing a syrup product from a starch-containing material, the method comprising the following steps: (a) liquefying a starch-containing material in the presence of α-amylase; (b) saccharifying the liquefied material in the presence of a variant glucosylamylase as described in any one of claims 1-9.

17. A method for producing a syrup product from a starch-containing material, the method comprising the following steps: In the presence of any of the variant glucosylamylases as described in any one of claims 1-10, the starch-containing material is saccharified at a temperature below the initial gelatinization temperature of the starch-containing material.

18. A polynucleotide encoding a variant as described in any one of claims 1-10.

19. A nucleic acid construct comprising the polynucleotide as described in claim 18.

20. An expression vector comprising the polynucleotide as described in claim 18.

21. A host cell comprising the polynucleotide of claim 18, the nucleic acid construct of claim 19, or the expression vector of claim 20.

22. The host cell according to claim 21, wherein the host cell is a yeast cell, specifically a yeast species, such as Saccharomyces cerevisiae.

23. The method of any one of claims 13-15, wherein the host cell of claim 22 is used as the fermentation organism in the fermentation step.

24. A method for producing a glucose amylase variant as described in any one of claims 1-10, the method comprising: The host cells as described in claim 21 are cultured under conditions suitable for expressing the variant; And optionally, the variants can be recycled.

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