α-amylase variants and their uses

Abstract

This technology provides a method to suppress the decrease in degradation efficiency due to α-amylase adsorption. [Solution] According to one embodiment of the present disclosure, an α-amylase mutant is provided. The α-amylase mutant has α-amylase activity and suppressed adsorption to a substrate, wherein at least one of the amino acid residues, aspartic acid or glutamic acid, on the surface of either a specific amino acid sequence or an amino acid sequence having 90% or more identity to said amino acid sequence, is replaced with at least one alanine or glycine.

Description

Technical Field

[0001] The present disclosure relates to α - amylase variants.

Background Art

[0002] In sake brewing, the α - amylase of Aspergillus oryzae contained in koji is a major enzyme that decomposes steamed rice and is an important enzyme related to the quality of sake. For example, as described in Non - Patent Document 1, conventionally, after the preparation of sake, it has been known that the decomposition efficiency decreases due to the adsorption of the α - amylase of Aspergillus oryzae to rice.

Prior Art Documents

Non - Patent Documents

[0003]

Non - Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] The decrease in decomposition efficiency due to the adsorption of α - amylase is a common problem not only in sake brewing but also in the decomposition of grains by α - amylase. Therefore, a technology capable of suppressing the decrease in decomposition efficiency due to the adsorption of α - amylase is required.

Means for Solving the Problems

[0005] The present disclosure can be realized in the following forms.

[0006] (1) According to one embodiment of the present disclosure, an α-amylase mutant is provided. In this α-amylase mutant, in either the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, at least one amino acid residue of aspartic acid or glutamic acid located on the surface of the three-dimensional structure is substituted with alanine or glycine, and the mutant has α-amylase activity and suppresses adsorption to the substrate. According to this form of α-amylase mutant, the decrease in degradation efficiency due to adsorption of α-amylase can be suppressed.

[0007] (2) In the α-amylase mutant described in (1) above, any of the following amino acid residues (A) to (F) may be substituted with alanine: (A) the amino acid residue at position 126 or equivalent of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid residue at position 133 or equivalent; (B) the amino acid residue at position 144 or equivalent of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid residue at position 156 or equivalent; and the amino acid residue at position 157 or equivalent; (C) the amino acid residue at position 177 or equivalent of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid residue at position 181 or equivalent; and the amino acid residue at position 186 or equivalent (D) The amino acid residue at the position corresponding to (SEQ ID NO: 1), and the amino acid residue at position 189 or the position corresponding to (SEQ ID NO: 1), (E) The amino acid residue at position 401 or the position corresponding to (SEQ ID NO: 1), and the amino acid residue at position 417 or the position corresponding to (F) The amino acid residue at position 401 or the position corresponding to (SEQ ID NO: 1), and the amino acid residue at position 417 or the position corresponding to (SEQ ID NO: 1). This form of α-amylase mutant can effectively suppress the decrease in degradation efficiency due to adsorption of α-amylase.

[0008] (3) In the α-amylase mutant described in (1) or (2) above, the amino acid residue of (C) above may be substituted with alanine. This form of α-amylase mutant can more effectively suppress the decrease in degradation efficiency due to adsorption of α-amylase.

[0009] (4) According to other forms of the present disclosure, a polynucleotide is provided which encodes an α-amylase variant as described in any one of (1) to (3) above. This form of polynucleotide makes it possible to express an α-amylase variant in which the reduction in degradation efficiency due to adsorption is suppressed.

[0010] (5) According to another form of the present disclosure, a vector comprising the polynucleotide described in (4) above is provided. With this form of vector, an α-amylase mutant in which the reduction in degradation efficiency due to adsorption is suppressed can be expressed.

[0011] (6) According to another form of the present disclosure, transformed cells comprising the vector described in (5) above are provided. These transformed cells express an α-amylase mutant in which the reduction in degradation efficiency due to adsorption is suppressed.

[0012] (7) According to another form of the present disclosure, Aspergillus oryzae is provided which expresses the α-amylase mutant described in any one of the above items (1) to (3). According to this form of Aspergillus oryzae, the decrease in degradation efficiency due to α-amylase adsorption can be suppressed.

[0013] (8) According to another form of the present disclosure, rice koji containing the koji mold described in (7) above is provided. With this form of rice koji, the decrease in decomposition efficiency due to α-amylase adsorption can be suppressed.

[0014] (9) Another embodiment of the present disclosure provides a method for producing sake. This method for producing sake includes the step of fermenting rice koji containing koji mold expressing an α-amylase mutant, steamed rice, and yeast, wherein the α-amylase mutant has at least one amino acid residue, either aspartic acid or glutamic acid, located on the surface of its three-dimensional structure, which is either the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and has α-amylase activity and suppressed adsorption to the substrate. According to this embodiment of the method for producing sake, the decrease in decomposition efficiency due to α-amylase adsorption can be suppressed, and therefore the decrease in the alcohol content can be suppressed.

[0015] (10) Another embodiment of the present disclosure provides a method for degrading cereals. This method for degrading cereals includes the step of degrading the cereals using an α-amylase mutant, wherein the α-amylase mutant has at least one amino acid residue, either aspartic acid or glutamic acid, located on the surface of its three-dimensional structure, substituted with alanine or glycine in either the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and possesses α-amylase activity while suppressing adsorption to a substrate. This embodiment of the method for degrading cereals suppresses the decrease in degradation efficiency due to α-amylase adsorption, and therefore suppresses a decrease in the degradation efficiency of cereals.

[0016] Furthermore, this disclosure can be implemented in various forms. For example, it can be implemented in the form of a method for producing α-amylase mutants, a method for producing koji mold, a method for producing rice koji, a method for producing moromi (fermented rice mash), a method for producing amazake (sweet rice wine), a method for producing mirin (sweet rice wine), a method for producing miso (soy sauce), a method for producing soy sauce, the use of α-amylase mutants for producing sake (rice wine), the use of α-amylase mutants for decomposing grains, a method for producing bioethanol, and so on. [Brief explanation of the drawing]

[0017] [Figure 1] It is an explanatory diagram showing the effects of pH and NaCl addition on the adsorption rate of steamed rice. [Figure 2] It is an explanatory diagram showing the results of SDS-PAGE in the culture supernatant of the medium. [Figure 3] It is an explanatory diagram showing the specific activity of the α-amylase mutant expressed in P. pastoris. [Figure 4] It is an explanatory diagram showing the adsorption rate of the α-amylase mutant expressed in P. pastoris to steamed rice. [Figure 5] It is an explanatory diagram showing the results of SDS-PAGE in the culture supernatant of the koji strain expressing the mutant α-amylase. [Figure 6] It is an explanatory diagram showing the specific activity of the α-amylase mutant expressed in Aspergillus oryzae. [Figure 7] It is an explanatory diagram showing the adsorption rate of the α-amylase mutant expressed in Aspergillus oryzae to steamed rice. [Figure 8] It is an explanatory diagram showing the state of the sake mash 12 hours after charging. [Figure 9] It is an explanatory diagram showing the change over time of the mash specific gravity of the sake mash. [Figure 10] It is an explanatory diagram showing the change over time of the solid content of the sake mash. [Figure 11] It is an explanatory diagram showing the change over time of the liquid content of the sake mash. [Figure 12] It is an explanatory diagram showing the change over time of the α-amylase activity in the liquid part of the sake mash.

Mode for Carrying Out the Invention

[0018] In this disclosure, "α-amylase" refers to an enzyme classified under EC3.2.1.1, also known as 1,4-α-D-glucan glucanohydrolase. α-amylase catalyzes the hydrolysis of starch and other linear or branched 1,4-glycosidoligosaccharides or polysaccharides. α-amylase activity can be determined by measuring the amount of reducing end produced by the enzymatic degradation of starch. More specifically, it can be measured, for example, using an α-amylase activity kit (manufactured by Kikkoman Biochemifa) according to the manufacturer's protocol. Examples of substrates for α-amylase include starch and other linear or branched 1,4-glycosidoligosaccharides or polysaccharides.

[0019] In this specification, "identity" of amino acid sequences or nucleotide sequences refers to the maximum degree of identity (%) of sequences obtained by aligning two sequences to be compared, introducing gaps as necessary. The identity of amino acid sequences or nucleotide sequences can be calculated, for example, using blastn from NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / ), which implements the BLAST algorithm.

[0020] In this disclosure, "amino acid residue" generally refers to the 20 amino acid residues that make up proteins, including alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0021] In this disclosure, amino acid residue substitutions may be represented using the IUPAC one-letter amino acid abbreviations as follows: [original amino acid, position, substituted amino acid]. In this case, for example, the substitution of aspartic acid at position 126 with alanine is indicated as "D126A". The substituted amino acid may also be omitted and represented as follows: [original amino acid, position]. In this case, for example, the substitution of aspartic acid at position 126 is indicated as "D126".

[0022] <α-amylase variant> According to one embodiment of the present disclosure, an α-amylase mutant is provided. In this α-amylase mutant, in either the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, at least one amino acid residue of aspartic acid or glutamic acid located on the surface of the three-dimensional structure is substituted with alanine or glycine, and it has α-amylase activity and suppresses adsorption to the substrate.

[0023] The amino acid sequence shown in Sequence ID No. 1 corresponds to the amino acid sequence of α-amylase derived from Aspergillus oryzae. In the following explanation, α-amylase having the amino acid sequence shown in Sequence ID No. 1, or α-amylase having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 1, will also be referred to as "parent α-amylase".

[0024] As an α-amylase having an amino acid sequence that has 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, it is more preferable that the identity with the amino acid sequence shown in SEQ ID NO: 1 is 92% or more, even more preferable that it is 94% or more, even more preferable that it is 95% or more, even more preferable that it is 96% or more, even more preferable that it is 97% or more, particularly preferable that it is 98% or more, and particularly preferable that it is 99% or more. The amino acid sequence having 90% or more identity includes an amino acid sequence in which one or more amino acid residues are deleted, inserted, substituted, or added. Examples of an "amino acid sequence in which one or more amino acid residues are deleted, inserted, substituted, or added" include an amino acid sequence in which one to 30, preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less amino acid residues are deleted, inserted, substituted, or added. It is preferable that the substitution of amino acid residues is a conservative substitution to an amino acid having a similar side chain.

[0025] Examples of conservative substitutions include substitutions between amino acid residues with basic side chains such as lysine, arginine, and histidine; substitutions between amino acid residues with acidic side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues with non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues with β-branched side chains such as threonine, valine, and isoleucine; and substitutions between amino acid residues with aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0026] In α-amylase mutants, the amino acid residues located on the surface of the three-dimensional structure can be determined using protein structure prediction software such as AlphaFold2. It is more preferable that two or more amino acid residues of at least one of aspartic acid and glutamic acid located on the surface of the three-dimensional structure are substituted, even more preferable that three or more are substituted, and even more preferable that four or more are substituted. Furthermore, it is even more preferable that at least one amino acid residue containing aspartic acid located on the surface of the three-dimensional structure is substituted. Also, it is more preferable that at least one amino acid residue of at least one of aspartic acid and glutamic acid located on the surface of the three-dimensional structure is substituted with alanine. Therefore, it is even more preferable that one or more aspartic acid residues located on the surface of the three-dimensional structure are substituted with alanine, even more preferable that two or more are substituted, particularly preferable that three or more are substituted, and especially most preferable that four or more are substituted. It is preferable that the amino acid residues in the active site of the α-amylase mutant are not substituted. The amino acid residues in the active site of α-amylase having the amino acid sequence shown in Sequence ID No. 1 include Asp206, Glu230, and Asp297.

[0027] Whether or not an α-amylase mutant possesses α-amylase activity can be determined by measuring the α-amylase activity according to the method described above. If the value is above the detection limit, it can be determined that the mutant possesses α-amylase activity. When measuring the α-amylase activity of an α-amylase mutant expressed in host cells such as Aspergillus oryzae, the culture itself or an extract from the culture can be used as a sample, either as is or concentrated, to measure the α-amylase activity. The α-amylase activity of the α-amylase mutant preferably shows a specific activity of 20% or more, more preferably 40% or more, even more preferably 60% or more, even more preferably 80% or more, even more preferably 100% or more, particularly preferably 120% or more, and particularly most preferably 140% or more.

[0028] The adsorption of the α-amylase mutant to the substrate can be confirmed by the following method. First, 10 units of enzyme solution and 2 g of substrate are reacted at 4°C for 12 hours. Then, the supernatant obtained by centrifugation at 3,000 rpm for 10 minutes is designated as supernatant a, and the precipitate is suspended in buffer and centrifuged again to obtain supernatant b. The amylase activity of supernatant a and supernatant b is measured, and the adsorption rate (%) can be calculated using the following formula. If the adsorption rate (%) is less than 80%, it can be determined that adsorption to the substrate is suppressed. The adsorption rate (%) of the α-amylase mutant to the substrate is preferably less than 70%, more preferably less than 60%, even more preferably less than 50%, even more preferably less than 40%, even more preferably less than 30%, particularly preferably less than 20%, and particularly most preferably less than 10%. Adsorption rate (%) = 100 - (Total activity of supernatant a + Total activity of supernatant b) / Enzyme activity added (10U) × 100

[0029] The α-amylase mutant is more preferably substituted with alanine or glycine in any of the following amino acid residues (A) to (F), more preferably substituted with alanine, and even more preferably substituted with amino acid residue (C). (A) The amino acid residue at position 126 or equivalent of the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue at position 133 or equivalent. (B) The amino acid residue at position 144 or the equivalent position of the amino acid sequence shown in Sequence ID No. 1, the amino acid residue at position 156 or the equivalent position, and the amino acid residue at position 157 or the equivalent position, (C) The amino acid residue at position 177 or equivalent of the amino acid sequence shown in Sequence ID No. 1, the amino acid residue at position 181 or equivalent, the amino acid residue at position 186 or equivalent, and the amino acid residue at position 189 or equivalent. (D) The amino acid residue at position 355 or equivalent of the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue at position 357 or equivalent. (E) The amino acid residue at position 401 or the equivalent position of the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue at position 417 or the equivalent position, (F) The amino acid residue at position 401 or equivalent of the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue at position 417 or equivalent.

[0030] The "corresponding position" on the amino acid sequence can be determined by aligning the target sequence and the reference sequence (the amino acid sequence shown in Sequence ID No. 1 in this disclosure) to give the greatest possible homology. Amino acid sequence alignment can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. Det. al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Clustal W can be used, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the alignment described above is considered to be the "corresponding position" to that arbitrary position.

[0031] The amino acid sequence alignment obtained above may be further fine-tuned for optimization. Such an optimal alignment is preferably determined by considering the similarity of the amino acid sequences and the frequency of inserted gaps. Here, similarity of amino acid sequences means the ratio (%) of the number of positions in which identical or similar amino acid residues exist in both sequences when two amino acid sequences are aligned, relative to the total number of amino acid residues. Similar amino acid residues refer to amino acid residues among the 20 amino acids that make up a protein that have similar properties in terms of polarity and charge, and that result in the aforementioned conservative substitutions.

[0032] The α-amylase variant of this disclosure suppresses the decrease in degradation efficiency due to α-amylase adsorption, thereby preventing a decrease in substrate degradation efficiency. As a result, the enzymatic reaction can proceed efficiently without raising the reaction temperature excessively. For this reason, the α-amylase variant of this disclosure is suitable for, for example, the production of sake.

[0033] <Polynucleotide encoding an α-amylase variant> The α-amylase variants of this disclosure can be produced using various mutagenesis techniques known in the art. For example, they can be produced by mutating a polynucleotide encoding the amino acid residue to be substituted in the parental α-amylase gene encoding the amino acid sequence of the parental α-amylase to a polynucleotide encoding the amino acid residue to be substituted, and then expressing the variant from that mutant gene. Thus, according to other forms of this disclosure, polynucleotides encoding the α-amylase variants of this disclosure are provided.

[0034] In this disclosure, various mutagenesis techniques known in the art can be used as means for mutating the amino acid residues of the parent α-amylase. For example, in a polynucleotide encoding the amino acid sequence of the parent α-amylase (hereinafter also referred to as the parent gene), the nucleotide sequence encoding the amino acid residue to be mutated can be changed to the nucleotide sequence encoding the amino acid residue after the mutation to obtain a polynucleotide encoding the mutant of the present invention. The introduction of the desired mutation into the parent gene can basically be carried out using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis methods can be carried out by any method, such as inverse PCR or annealing. Commercially available site-directed mutagenesis kits (for example, Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit, etc.) can also be used.

[0035] Site-directed mutagenesis into a parent gene can most commonly be performed using a mutation primer containing the nucleotide mutation to be introduced. This mutation primer should be designed to anneal to a region in the parent gene containing the nucleotide sequence encoding the amino acid residue to be mutated, and to include a nucleotide sequence (codon) that encodes the mutated amino acid residue in place of the nucleotide sequence (codon) encoding the mutated amino acid residue. Those skilled in the art can appropriately recognize and select the nucleotide sequences (codons) encoding the pre-mutation and post-mutation amino acid residues. Alternatively, site-directed mutagenesis can also be performed using a method in which two complementary primers containing the nucleotide mutation to be introduced are used separately to amplify the upstream and downstream sides of the mutation site, and the resulting DNA fragments are then ligated together by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): p61-68).

[0036] Template DNA containing the parent gene can be prepared by extracting genomic DNA from a microorganism producing the parent α-amylase according to conventional methods, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, the corresponding nucleotide sequence may be chemically synthesized based on the amino acid sequence of the parent α-amylase and used as template DNA.

[0037] Mutation primers can be prepared by well-known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic Acids R4esearch, 1989, 17:7059-7071). Such primer synthesis can also be carried out using commercially available oligonucleotide synthesizers (e.g., ABI). By using a primer set containing these mutation primers and introducing site-directed mutagenesis as described above with the parental gene as template DNA, polynucleotides encoding the mutant of the present invention having the desired mutation can be obtained.

[0038] The polynucleotides encoding the α-amylase variants of this disclosure may include single-stranded or double-stranded DNA, cDNA, RNA, or other artificial nucleic acids. The polynucleotides may also be chemically synthesized. Furthermore, the polynucleotides may include nucleotide sequences of the untranslated region (UTR) in addition to the open reading frame (ORF). The polynucleotides may also be codon-optimized to suit the species of transformant for producing the α-amylase variants of this disclosure. Information on codons used by various organisms is available, for example, from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0039] <Vector containing polynucleotides encoding α-amylase variants> The polynucleotides encoding the α-amylase variants of this disclosure can be incorporated into a vector. Thus, according to other forms of this disclosure, a vector comprising the polynucleotides encoding the α-amylase variants of this disclosure is provided.

[0040] The type of vector containing the polynucleotide is not particularly limited, and examples include plasmids, phages, phagemids, cosmids, viruses, YAC vectors, and shuttle vectors. Furthermore, the vector is not particularly limited, but it is more preferably a vector that can be amplified within bacteria, preferably Bacillus bacteria (e.g., Bacillus subtilis or its mutants), and even more preferably an expression vector that can induce the expression of a transgene within Bacillus bacteria. In particular, a shuttle vector, which is a vector that can replicate in either Bacillus bacteria or other organisms, can be suitably used for recombinant production of the α-amylase variant of this disclosure.

[0041] The above vector may include a DNA region containing a DNA replication initiation region or origin of replication. Alternatively, in the above vector, a regulatory sequence such as a promoter region, a terminator region, or a secretion signal region for secreting the expressed protein extracellularly may be operably linked upstream of the polynucleotide encoding the α-amylase variant of this disclosure (i.e., the mutant gene). "Operatally linked" means that the gene and the regulatory sequence are positioned such that the gene can be expressed under the control of the regulatory region. The types of regulatory sequences such as promoter regions, terminators, and secretion signal regions are not particularly limited, and commonly used promoters and secretion signal sequences can be appropriately selected and used depending on the host to which the gene is introduced. For example, suitable examples of regulatory sequences that can be incorporated into a vector include the promoter and secretion signal sequence of the cellulase gene of Bacllus sp. KSM-S237 strain.

[0042] Furthermore, the above vector may also incorporate marker genes (for example, resistance genes for drugs such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Additionally, when using a nutrient-requiring strain as a host, a gene encoding an enzyme for synthesizing the required nutrients may be incorporated into the vector as a marker gene. Furthermore, when using a selective medium that requires specific metabolism for growth, a gene related to that metabolism may be incorporated into the vector as a marker gene. The linking of the above polynucleotides with the regulatory sequence and marker genes can be performed by methods known in the field, such as SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68).

[0043] <Transformed cells> Transformed cells can be obtained by introducing a vector containing a polynucleotide encoding the α-amylase variant of the present disclosure into a host. Accordingly, according to another form of the present disclosure, transformed cells comprising a vector containing a polynucleotide encoding the α-amylase variant of the present disclosure are provided.

[0044] The host cell is not particularly limited and includes microorganisms such as filamentous fungi, yeasts, and bacteria. Examples of filamentous fungi include Aspergillus, Trichoderma, and Rhizopus. Examples of yeasts include Saccharomyces cerevisiae and Pichia pastoris. Examples of bacteria include Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus. The host cell is preferably a filamentous fungus, and more preferably Aspergillus oryzae. The koji mold used is not particularly limited and includes, for example, yellow koji mold (Aspergillus oryzae), white koji mold (Aspergillus Kawachii), black koji mold (Aspergillus luchuensis), red koji mold (Monascus purpureus), and dried koji mold (Eurotium herbariorum), but yellow koji mold (Aspergillus oryzae) is even more preferable.

[0045] The method for introducing the vector into the host is not particularly limited; for example, methods commonly used in the field, such as the protoplast method or electroporation, can be used. By selecting strains in which the introduction has been properly carried out based on indicators such as marker gene expression and nutritional requirements, the desired transformant into which the vector has been introduced can be obtained.

[0046] By culturing a transformant into which a vector containing a polynucleotide encoding the α-amylase variant of this disclosure has been introduced in an appropriate medium, the gene encoding the protein on the vector will be expressed, and as a result, the α-amylase variant of this disclosure can be expressed. The medium used to culture the transformant can be appropriately selected according to the type of microorganism of the transformant. Furthermore, the α-amylase variant of this disclosure may be expressed from the polynucleotide encoding the α-amylase variant of this disclosure or its transcript by using a cell-free translation system. A "cell-free translation system" is an in vitro transcription-translation system or in vitro translation system constructed by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0047] The α-amylase variants of this disclosure, generated in the culture or cell-free translation system described above, may be isolated or purified by common protein purification methods, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combinations. Furthermore, if the gene encoding the α-amylase variant of this disclosure and the secretion signal sequence are operably linked on a vector within the transformant, the resulting protein is secreted extracellularly and can be more easily recovered from the culture. The protein recovered from the culture may be further purified by known means.

[0048] <Koji mold and rice koji> In other forms of this disclosure, koji mold expressing the α-amylase mutant of this disclosure is provided. The koji mold is not particularly limited and includes, for example, yellow koji mold (Aspergillus oryzae), white koji mold (Aspergillus Kawachii), black koji mold (Aspergillus luchuensis), red koji mold (Monascus purpureus), and dried koji mold (Eurotium herbariorum), but yellow koji mold (Aspergillus oryzae) is more preferred. By using yellow koji mold expressing the α-amylase mutant of this disclosure, the decrease in decomposition efficiency due to α-amylase adsorption can be suppressed, and thus the decrease in decomposition efficiency of steamed rice, etc. can be suppressed. For this reason, it is suitable for the production of sake, shochu, amazake, mirin, miso, soy sauce, etc., and is particularly suitable for the production of sake.

[0049] Another form of this disclosure provides rice koji containing Aspergillus oryzae expressing the α-amylase variant of this disclosure. This rice koji is produced by inoculating steamed rice with Aspergillus oryzae expressing the α-amylase variant of this disclosure and then allowing it to propagate for a predetermined time. The rice used in the production of rice koji is not particularly limited and may be polished rice of any milling ratio or brown rice, but polished rice is preferred. The form of the rice used in the production of rice koji is not particularly limited, but examples include whole rice, broken rice, powder, etc. The temperature, humidity, moisture content, culture time, etc. in the production of rice koji can be set as appropriate. With the rice koji of this disclosure, the decrease in decomposition efficiency due to α-amylase adsorption can be suppressed, and therefore the decrease in decomposition efficiency of steamed rice, etc. can be suppressed. For this reason, it is suitable for the production of sake, shochu, amazake, mirin, miso, soy sauce, etc., and is particularly suitable for the production of sake.

[0050] <Method of producing sake> Other forms of this disclosure provide a method for producing sake. In this disclosure, "sake" means a fermented product made primarily from rice, rice koji, yeast, and water, and preferably means sake as defined in the Liquor Tax Law of Japan as of the filing date of this application.

[0051] The sake production method of this disclosure includes a step of fermentation using the above-mentioned rice koji containing Aspergillus oryzae expressing the α-amylase mutant of this disclosure, steamed rice, and yeast. The fermentation step can also be rephrased as a mash production step. The steamed rice used in the fermentation step is not particularly limited and may be steamed rice polished to any degree, or it may be steamed brown rice, but it is preferable that it be steamed polished rice. The yeast used in the fermentation step is not particularly limited, but it is preferable that it includes Saccharomyces cerevisiae. It is preferable that the yeast used in the fermentation step is in a form included in the sake starter. The conditions such as culture temperature and culture time in the fermentation step can be set as appropriate. The sake production method may include a koji-making step prior to the fermentation step, in which Aspergillus oryzae is propagated on rice to produce rice koji. Furthermore, the sake manufacturing method may include a pressing step in which, after the fermentation process, the liquid fraction and sake lees are separated from the resulting mash and the liquid fraction is recovered, and a processing step in which one or more of the following treatments are carried out on the recovered liquid fraction: heat treatment, sediment removal, and filtration.

[0052] This method of sake production suppresses the decrease in decomposition efficiency due to α-amylase adsorption, thereby preventing a decrease in the decomposition efficiency of steamed rice. As a result, the amount of alcohol obtained relative to the mass of raw rice can be increased, thus preventing a decrease in the alcoholization rate. Furthermore, even when using rice that is difficult to decompose in sake mash, such as high-temperature damaged rice, the rice can be decomposed before it ages, thus preventing a decrease in the alcoholization rate. In addition, the sake production method of this disclosure suppresses the decrease in the decomposition efficiency of steamed rice, allowing the enzymatic reaction to proceed efficiently without excessively high fermentation temperatures. As a result, energy efficiency in plants and other facilities can be improved, and the efficiency of material production can be increased. Moreover, by suppressing the decrease in decomposition efficiency due to α-amylase adsorption, the viscosity of the mash can be reduced, making stirring easier and increasing the uniformity of the mash.

[0053] <Methods for breaking down grains> Other forms of this disclosure provide a method for degrading cereals. The method for degrading cereals of this disclosure includes the step of degrading the cereals using the α-amylase variants of this disclosure. Cereals are not particularly limited in that they include those that can serve as substrates for α-amylase, but examples include cereals of the Poaceae, Polygonaceae, Amaranthaceae, and Cyperaceae families. Cereals belonging to the Poaceae family are not particularly limited, but examples include rice, wheat, barley, maize, oats, millet, sorghum, and barnyard millet. Cereals belonging to the Polygonaceae family are not particularly limited, but examples include buckwheat. Cereals belonging to the Amaranthaceae family are not particularly limited, but examples include foxtail millet. Cereals belonging to the Cyperaceae family are not particularly limited, but examples include teff. In this disclosure, "cereals" includes potatoes and cassava.

[0054] The process of decomposing grains can be carried out as follows, for example: The raw material grains are crushed and water is added to form a slurry. Then, the α-amylase mutant of this disclosure is added to proceed with the decomposition. The reaction temperature, reaction time, and other conditions in the process of decomposing grains can be set as appropriate. In addition to the α-amylase mutant of this disclosure, other enzymes such as glucoamylase and amyloglucosidase may also be used in combination in the process of decomposing grains. The decomposition product obtained by the method of decomposing grains of this disclosure may be fermented by adding yeast or the like as needed, and the ethanol from the fermentation liquid may be recovered as bioethanol by distillation and dehydration. Furthermore, the method of decomposing grains of this disclosure can suppress the decrease in decomposition efficiency due to α-amylase adsorption, and as a result, the enzymatic reaction can proceed efficiently without raising the fermentation temperature excessively. As a result, energy efficiency can be improved in plants, etc., and the efficiency of material production can be improved. [Examples]

[0055] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0056] <Experiment 1> 1. Method (1) Adsorption test The adsorption rate of α-amylase to steamed rice was determined by the following method. Steamed rice was prepared by soaking 65% polished Yamada Nishiki rice for 6 hours, draining it for 1.5 hours, and then steaming it for 30 minutes. Enzyme solution (10 units), lactate buffer (25 mM, pH 4.0), and 2 g of steamed rice were mixed in a 15 ml sample tube and allowed to stand overnight at 4°C. The mixture was then centrifuged at 3,000 rpm for 10 minutes, and the supernatant was designated as supernatant a. 5 ml of 25 mM lactate buffer (pH 4.0) was added to the precipitate obtained by centrifugation to suspend it, and the supernatant obtained by centrifugation again was designated as supernatant b. The α-amylase activity in supernatant a and supernatant b was measured, and the adsorption rate (%) was calculated based on the formula described above. In addition, the adsorption rates were similarly determined when a lactate buffer (25mM, pH 6.0) was used instead of the above-mentioned lactate buffer (25mM, pH 4.0), and when a lactate buffer (25mM, pH 4.0) containing 1% NaCl was used.

[0057] (2) Production of α-amylase expression plasmid using Pichia pastoris Aspergillus oryzae RIB40 (provided by the National Research Institute of Brewing), a wild strain of Aspergillus oryzae, was inoculated into YPD medium (Yeast Extract 1%, Peptone 0.5%, Glucose 1%) and cultured overnight at 30°C and 180 rpm with shaking. The mycelium was collected by filtration with Miracrose (Merck Millipore), washed with sterile water, immediately frozen with liquid nitrogen, and crushed using a mortar and pestle. Total RNA was extracted using ISOGEN (Nippon Gene) according to the protocol. Total RNA treated with DNase I (Takara Bio) at 37°C for 30 minutes was purified by the phenol-chloroform method, and cDNA was synthesized using Superscript III (Thermo Fisher Scientific) according to the protocol. Using the obtained cDNA as a template, amyA cDNA was obtained by PCR using KOD-Plus-Neo (TOYOBO). As primers, amyAInFfw:5'-TGAAGCTATGATGGTCGCGTGGTGGTC-3' (shown in SEQ ID NO: 2) and amyAInFrv:5'-ACCATCATAGCTTCAGCCTCTCTTTTCT-3' (shown in SEQ ID NO: 3) were used. PCR was performed at 94°C for 2 minutes, followed by 30 cycles of 98°C for 10 seconds and 68°C for 45 seconds. Plasmid pPAMO was obtained by in-fusion cloning of amyA cDNA to linearized pPICZαA (Thermo Fisher Scientific) using the In-Fusion® HD Cloning Kit (TaKaRa) according to the protocol. For linearization of pPICZαA, PICamyAInFfw:5'-TTTTTGTTCACGAGCTACTACAGATCTTG-3' (SEQ ID NO: 4) and PICamyAInFrv:5'-GCTCGTGAACAAAAACTCATCTCAGAAG-3' (SEQ ID NO: 5) were used, and PCR was performed under the conditions of 94°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 2 minutes for 30 cycles. pPAMO contains secretion signals derived from amyA and secretion signals from α-factor contained in pPICZαA.Therefore, in order to remove the secretion signal derived from amyA, PCR was performed using pPAMO as a template with the primers no-signalFw:5'-GCAACGCCTGCGGACTGGC-3' shown in SEQ ID NO. 6 and no-signalRv:5'-GTCCGCAGGCGTTGCAGCTTCAGCCTCTCTTTTCT-3' shown in SEQ ID NO. 7, under conditions of 94°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 2 minutes for 30 cycles. Plasmid pPAMO_M-1, from which the secretion signal derived from amyA had been removed, was obtained by in fusion cloning of the resulting PCR product.

[0058] (3) Preparation of alanine substitution plasmids Using pPAMO_M-1 as a template, expression plasmids of mutant α-amylase in which the target amino acid was substituted with alanine were constructed by PCR and intramolecular in-fusion cloning using a primer set containing the target mutation. The amino acid substituted with alanine is shown in Table 1 below, and the primers used for PCR, along with their sequence numbers, are shown in Table 2 below. PCR was performed under the following conditions: 94°C for 2 minutes, followed by 98°C for 10 seconds, 76°C for 30 seconds, and 68°C for 50 seconds for 30 cycles. The resulting plasmids were designated pPAMO_1 to pPAMO_17.

[0059] [Table 1]

[0060] [Table 2]

[0061] (4) Yeast transformation Pichia pastoris-X33 strain (Thermo Fisher Scientific) was used as the parent strain and transformed by electroporation. P. pastoris-X33 was dispensed into a 50 ml sample tube and cultured overnight in 5 ml of YPD liquid medium (Yeast Extract 1%, Peptone 2%, Glucose 2%) at 30°C. 0.1 ml of this culture was added to 100 ml of YPD liquid medium (Yeast Extract 1%, Peptone 2%, Glucose 2%) in a 300 ml Erlenmeyer flask and cultured at 30°C until the OD600 reached 1.3-1.5. pPAMO_M-1 and pPAMO_1-17 were linearized by digestion with the restriction enzyme SacI (Takara Bio), and introduced into P. pastoris-X33 using electroporation with an electroporator (Bio-Rad). For recovery culture, YPDS medium (Peptone 2%, Glucose 2%, Yeast Extract 1%, Sorbitol 18.2%, Agar 2%, Zeocin 100 μg / mL) was used, and the cultures were incubated at 30°C for 6 days. The resulting strains were designated as the target transformants. The obtained pPAMO_1~17-transformed strains were designated as P. pastoris AMO1~17 strains, respectively.

[0062] (5) Expression of α-amylase by P. pastoris Using a 300 ml baffled flask, 30 mL of BMGY medium (Yeast Extract 1%, Peptone 2%, Glycerol 1%, Yeast Nitrogen Base 1.34%, 1M Potassium Phosphate Buffer pH 6.0, 100 ml, Biotin 0.4 ppm, DW up to 1000 ml) was incubated at 28.5°C for 24 hours. After incubation, the mixture was centrifuged at 2000 rpm for 5 minutes in a 50 mL sample tube. The supernatant was then removed, the precipitated bacterial cells were suspended in approximately 5 ml of sterile water, and centrifuged again at 2000 rpm for 5 minutes. The supernatant was then discarded. This process was repeated twice, and the resulting bacterial cells were cultured in 30 ml of BMMY medium (Yeast Extract 1%, Peptone 2%, Methanol 0.5%, Yeast Nitrogen Base 1.34%, 100 ml of 1 M Potassium Phosphate Buffer pH 6.0, Biotin 0.4 ppm, DW up to 1000 ml) at 28.5°C for 72 hours. During this time, 0.5% methanol was added to the medium every 24 hours. After the culture was complete, the bacterial cells were removed by centrifugation, and the supernatant was collected after filter sterilization using DISMIC®-25CS045AS (manufactured by ADVANTEC), and this was used as the α-amylase enzyme solution.

[0063] (6) SDS-PAGE The analytical samples were concentrated by ultrafiltration using Centrifugal Filters Ultracel®-30K (Sigma Aldrich), and the culture medium components were replaced with 25 mM lactate buffer (pH 4.0). For acetone precipitation, the sample was mixed with four times the volume of cold acetone, allowed to stand overnight at -20°C, then centrifuged at 13,000 rpm for 5 minutes, and suspended in Sample Buffer (Tris-HCl 0.125 M, 2-Mercaptoethanol 10%, SDS 4%, Sucrose 10%, Bromophenol blue 40 ppm). This was boiled at 100°C for 5 minutes and used as the sample. For SDS-PAGE, a 10% polyacrylamide gel was used and 10 μl of the sample was loaded. After electrophoresis, staining was performed using CBB R-250. Protein amounts were quantified by the signal intensity of SDS-PAGE. CBB-stained gel images were imported into ImageJ, the signal intensity was quantified, and used for comparison.

[0064] (7) Measurement of enzyme activity The α-amylase activity of each sample was measured using the α-amylase activity kit (Kikkoman Biochemifa Co., Ltd.), and the acid protease activity was measured using the acid protease measurement kit (Peptide Laboratories, Inc.) and the saccharification fractional quantification kit (Kikkoman Biochemifa Co., Ltd.). For the enzyme activity measurement of rice koji, 5g of prepared rice koji was immersed in 50ml of 10mM acetate buffer + 0.5% NaCl (pH 5.0), left to stand overnight at 4°C, filtered with Miracrose (Millipore), and then filtered with DISMIC®-25CS045AS (ADVANTEC) to obtain the sample. In the small-scale fermentation test, specific gravity was measured using Easy Dens (Anton Paar), alcohol concentration was measured using Alcomate (Riken Keiki Co., Ltd.), and acidity and amino acid content were analyzed according to the analytical methods prescribed by the National Tax Agency. First, the enzyme solution was prepared. For the TAA mutant prepared with P. pastoris, the BMMY culture supernatant concentrate was used as the enzyme solution. For the α-amylase mutant prepared with A. oryzae, the rice koji extract prepared with 25 mM lactate buffer (pH 4.0) was used as the enzyme solution.

[0065] 2.Results (1) Construction of the adsorption test system and confirmation of the adsorption rate Figure 1 is an explanatory diagram showing the effects of pH and NaCl addition on the adsorption rate of steamed rice. As described above, 10 U of α-amylase was added to a buffer solution containing steamed rice, and the adsorption rate was determined by subtracting the α-amylase activity observed in the supernatant after centrifugation for a certain period of time. When adsorption tests were performed using rice koji extract, the adsorption rate of α-amylase was over 90% at pH 4.0, which has been reported to be a good adsorption rate between steamed rice and koji mold α-amylase. However, under pH 6.0, which is said to decrease the adsorption rate, the adsorption rate decreased to about 40%. Furthermore, under the condition of adding 1% NaCl, which is said to cause almost no adsorption, the adsorption rate decreased to about 20%. Therefore, since the results obtained generally reflect the known adsorption trend between steamed rice and koji mold α-amylase, this test system was considered appropriate, and the pH 4.0 condition was used for evaluating the α-amylase adsorption rate in subsequent experiments.

[0066] (2) Preparation and evaluation of α-amylase mutants using P. pastoris α-amylase mutants were created by partially substituting acidic amino acids on the surface of the α-amylase structure of A. oryzae. Using AlphaFold2, a protein structure prediction software, the acidic amino acids on the surface of the parent α-amylase structure were predicted. As a result, 47 acidic amino acids were predicted, including Asp206, Glu230, and Asp297 in the active site. These were targeted and synthesized by locally substituting Ala using PCR-based site-specific mutagenesis. Seventeen α-amylase mutants (No. 1-17) were designed, as shown in Table 1 above. Plasmids for expression in P. pastoris were prepared and introduced into the P. pastoris-X33 strain. Protein expression was induced using methanol in the resulting transformants, and the amylase activity of the culture medium supernatant was measured. The measurement results are shown in Table 3 below.

[0067] [Table 3]

[0068] As shown in Table 3, under the conditions of this example, α-amylase activity was confirmed in P. pastoris mutant α-amylase-expressing strains No. 5, 6, 8, 14, 16, and 17. It should be noted that under lower temperatures, longer culture times, or other conditions, other P. pastoris mutant α-amylase-expressing strains may also exhibit α-amylase activity.

[0069] Figure 2 is an explanatory diagram showing the results of SDS-PAGE in the culture medium supernatant. In Figure 2, the test samples are P. pastoris mutant α-amylase expression strains No. 5, 6, 8, 14, 16, and 17, along with the control wild-type α-amylase expression strain (P. pastoris_M-1 A1). As shown in Figure 2, a clear band was observed in all samples around the molecular weight of 53 kDa, which is the size of A. oryzae α-amylase. The specific activity of each sample was determined by calculating the relative protein amount from the intensity of the band in the CBB staining of the SDS-PAGE. Furthermore, the adsorption rate of each α-amylase mutant to steamed rice was determined using the method described above.

[0070] Figure 3 is an explanatory diagram showing the specific activity of α-amylase mutants expressed in P. pastoris. As shown in Figure 3, no significant decrease in specific activity was observed compared to the unmutated wild-type α-amylase (WT).

[0071] Figure 4 is an explanatory diagram showing the adsorption rate of α-amylase mutants expressed in P. pastoris to steamed rice. As shown in Figure 4, when each α-amylase mutant was subjected to an adsorption test, the adsorption rate decreased in all α-amylase mutants. In particular, strain No. 8 expressing the P. pastoris mutant α-amylase showed the lowest adsorption rate, at approximately 35%. These results indicate that multiple acidic amino acids are involved in the adsorption of α-amylase to steamed rice. Since the adsorption rate of non-mutated α-amylase (WT) is approximately 90%, the α-amylase activity of strain No. 8, which has an adsorption rate of approximately 35%, is expected to increase 6 to 7 times compared to the wild type.

[0072] <Experiment 2> (1) Preparation and evaluation of α-amylase mutants using A. oryzae To eliminate the influence of α-amylase derived from Aspergillus oryzae genomic DNA, the parent strain used for Aspergillus oryzae transformation was the A. oryzaeΔamyABC strain (Yokota, Ji., Shiro, D., Tanaka, M. et al. Cellular responses to the expression of unstable secretory proteins in the filamentous fungus Aspergillus oryzae. Appl Microbiol Biotechnol 101, 2437-2446 (2017)), which lacks the α-amylase gene. A strain expressing the gene expressed in the P. pastoris mutant α-amylase-expressing strain was created under a high-expression promoter. As a control, a strain expressing α-amylase without the mutation was also created. The obtained mutant α-amylase-expressing Aspergillus oryzae strains No. 5, 6, 8, 14, 16, and 17, and the control α-amylase-expressing strain were each cultured in liquid culture in YPM medium at 30°C for 5 days.

[0073] Figure 5 is an explanatory diagram showing the results of SDS-PAGE in the culture supernatant of mutant α-amylase-expressing Aspergillus oryzae strains. In Figure 5, the test samples are A. oryzae mutant α-amylase-expressing strains No. 5, 6, 8, 14, 16, and 17, along with a control strain expressing α-amylase without the mutation. As shown in Figure 5, a clear band was observed in all samples around the molecular weight of 53 kDa, which is the size of A. oryzae α-amylase. The specific activity of each sample was determined by calculating the relative protein amount from the intensity of the band in the CBB staining of the SDS-PAGE. In addition, rice koji was prepared using these Aspergillus oryzae strains, and an adsorption test was performed on the α-amylase obtained as a rice koji extract.

[0074] Figure 6 is an explanatory diagram showing the specific activity of α-amylase mutants expressed in Aspergillus oryzae. As shown in Figure 6, no mutants were found to have significantly reduced specific activity compared to the non-mutated α-amylase (Control).

[0075] Figure 7 is an explanatory diagram showing the adsorption rate of α-amylase mutants expressed in Aspergillus oryzae to steamed rice. As shown in Figure 7, the adsorption rate decreased in all α-amylase mutants compared to the control. This result is similar to the adsorption test results of α-amylase mutants expressed in P. pastoris shown in Figure 4. Among the A. oryzae mutant α-amylase expressing strains, No. 8 had the lowest adsorption rate, at approximately 39%.

[0076] (2) Small-scale sake brewing experiment using rice koji expressing α-amylase mutant Rice koji was prepared using three strains: Aspergillus oryzae strain No. 8, which expresses a mutant α-amylase with significantly reduced adsorption rate; Aspergillus oryzae strain No. 8, which expresses α-amylase without the mutation (control strain); and A. oryzae RIB40, a genome-analyzed strain of A. oryzae. The enzyme activities of the prepared rice koji were measured. The measurement results are shown in Table 4 below.

[0077] [Table 4]

[0078] As shown in Table 4, the α-amylase activity of the prepared rice koji was approximately 740-830 units / g·koji lower in strain No. 8 and the control strain compared to the α-amylase activity of strain RIB40. This result is thought to be due to the fact that both strain No. 8 and the control strain have only one copy of the α-amylase gene.

[0079] Using the three rice koji strains mentioned above, water, steamed rice, rice koji, yeast, and lactic acid were mixed to produce sake on a small scale. The change in the specific gravity of the mash over time after brewing (after the start of the experiment) was compared in Baumé degrees, and the amount of solid and liquid parts of the sake mash was also compared over time. In addition, the α-amylase activity (Unit / ml) contained in the sample supernatant was measured at 12, 20, and 36 hours after the start of the experiment.

[0080] Figure 8 is an explanatory diagram showing the state of sake mash 12 hours after brewing. As shown in Figure 8, the sake mash using mutant α-amylase-expressing Aspergillus oryzae strain No. 8 showed clear dissolution of steamed rice at 12 hours after brewing, compared to sake mash using the control strain and RIB40 strain.

[0081] Figure 9 is an explanatory diagram showing the change in the specific gravity of sake mash over time. In Figure 9, the horizontal axis represents the number of days elapsed since brewing, and the vertical axis represents the degree of Baumé (Be). As shown in Figure 9, the specific gravity of the sake mash using mutant α-amylase-expressing Aspergillus oryzae strain No. 8 remained high for the first two days immediately after brewing. This result was thought to be due to the fact that the dissolution of steamed rice progressed immediately after brewing, and the specific gravity of the liquid portion of the sake mash increased due to the sugars produced by the dissolution.

[0082] Figure 10 is an explanatory diagram showing the change in the solid content of sake mash over time. Figure 11 is an explanatory diagram showing the change in the liquid content of sake mash over time. In Figures 10 and 11, the horizontal axis represents the number of days elapsed since brewing, and the vertical axis represents the solid content (g) or liquid content (g). As shown in Figures 10 and 11, in sake mash using mutant α-amylase-expressing Aspergillus oryzae strain No. 8, it was confirmed that the solid content decreased rapidly and the liquid content increased during the first two days after brewing due to the dissolution of steamed rice.

[0083] Figure 12 is an explanatory diagram showing the change in α-amylase activity over time in the liquid portion of sake mash. In Figure 12, the horizontal axis represents the elapsed time (hr) since brewing, and the vertical axis represents the α-amylase activity (Unit / ml). As shown in Figure 12, the sake mash using mutant α-amylase-expressing Aspergillus oryzae strain No. 8 showed high α-amylase activity immediately after brewing, and it was confirmed that the α-amylase activity in the liquid portion was high because the adsorption of α-amylase to steamed rice was suppressed. From these results, it was confirmed that mutant α-amylase No. 8 has the characteristic of being able to rapidly dissolve steamed rice by suppressing adsorption to steamed rice through amino acid substitution.

[0084] The present invention is not limited to the embodiments described above, and can be realized in various configurations without departing from its spirit. For example, the technical features in the embodiments and examples corresponding to the technical features in each form described in the summary of the invention can be replaced or combined as appropriate in order to solve some or all of the above-described problems, or to achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. It is an α-amylase mutant, In either the amino acid sequence shown in Sequence ID No. 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 1, at least one amino acid residue of aspartic acid or glutamic acid located on the surface of the three-dimensional structure is substituted with alanine or glycine. It possesses α-amylase activity and has suppressed adsorption to the substrate. α-amylase mutant.

2. In the α-amylase mutant described in claim 1, One of the amino acid residues (A) to (F) below is substituted with alanine. α-amylase mutant: (A) The amino acid residue at position 126 or a corresponding position in the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue at position 133 or a corresponding position, (B) The amino acid residue at position 144 or the equivalent position of the amino acid sequence shown in Sequence ID No. 1, the amino acid residue at position 156 or the equivalent position, and the amino acid residue at position 157 or the equivalent position (C) The amino acid residue at position 177 or the corresponding position of the amino acid sequence shown in Sequence ID No. 1, the amino acid residue at position 181 or the corresponding position, the amino acid residue at position 186 or the corresponding position, and the amino acid residue at position 189 or the corresponding position (D) The amino acid residue at position 355 or equivalent of the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue at position 357 or equivalent. (E) The amino acid residue at position 401 or a corresponding position in the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue at position 417 or a corresponding position, (F) The amino acid residue at position 401 or a corresponding position in the amino acid sequence shown in Sequence ID No. 1, and the amino acid residue at position 417 or a corresponding position.

3. In the α-amylase mutant described in claim 1, The amino acid residue in (C) above is substituted with alanine. α-amylase mutant.

4. A polynucleotide encoding an α-amylase variant according to any one of claims 1 to 3.

5. A vector comprising the polynucleotide described in claim 4.

6. A transformed cell comprising the vector according to claim 5.

7. Aspergillus oryzae expressing the α-amylase mutant described in any one of claims 1 to 3.

8. Rice koji containing the koji mold described in claim 7.

9. A method for producing sake, The process includes a step of fermentation using rice koji containing Aspergillus oryzae that expresses an α-amylase mutant, steamed rice, and yeast. The aforementioned α-amylase mutant is In either the amino acid sequence shown in Sequence ID No. 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 1, at least one amino acid residue of aspartic acid or glutamic acid located on the surface of the three-dimensional structure is substituted with alanine or glycine. It possesses α-amylase activity and has suppressed adsorption to the substrate. The method of producing sake.

10. A method for decomposing grains, The process includes a step of decomposing the aforementioned grains using an α-amylase variant, The aforementioned α-amylase mutant is In either the amino acid sequence shown in Sequence ID No. 1 or an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 1, at least one amino acid residue of aspartic acid or glutamic acid located on the surface of the three-dimensional structure is substituted with alanine or glycine. It possesses α-amylase activity and has suppressed adsorption to the substrate. Methods for breaking down grains.

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