Modified transglucosidase

Abstract

The purpose of the present invention is to provide, for example, transglucosidase having modified reaction specificity and a method of using the modified transglucosidase to produce a composition containing an enzyme agent and a carbohydrate. The above problem is addressed by providing a modified transglucosidase that is constituted by, for example, a polypeptide which is constituted by an amino acid sequence obtained by introducing, into an amino acid sequence shown by SEQ ID NO: 1 or 13, at least one of (A) substitution of an amino acid residue at position 227 with a leucine residue or a phenylalanine residue, (B) substitution of an amino acid residue at position 343 with a tyrosine residue, and (C) substitution of an amino acid residue at position 493 with a valine residue.

Description

Modified transglucosidase

[0001] The present invention relates to a transglucosidase with modified reaction specificity, and also to the use of the transglucosidase in various applications.

[0002] Transglucosidase (α-glucosidase) is an enzyme that primarily hydrolyzes the α-1,4-glucosidic bond at the non-reducing end of maltooligosaccharides. However, at high substrate concentrations, it also catalyzes the transglycosylation activity to produce isomaltooligosaccharides with α-1,6-glucosidic bonds. Transglucosidase is used to produce isomaltooligosaccharides from maltose, a product of starch degradation, and to improve the flavor of alcoholic beverages by producing oligosaccharides.

[0003] Various reports have been published so far regarding the modification of transglucosidase.

[0004] For example, Patent Document 1 describes that by introducing a mutation into transglucosidase derived from Dizosaccharomyces pombe, the hydrolytic activity can be reduced to 1 / 10,000 or less.

[0005] Patent Document 2 describes that the transglycosylation activity of an anomer-retaining glycosidase can be improved by substituting the aspartic acid or glutamic acid residue of the nucleophilic catalytic residue with a cysteine ​​sulfinic acid residue and / or a cysteine ​​sulfonic acid residue.

[0006] Patent Document 3 describes a method for converting dextran glucosidase derived from Streptococcus mutans into a protein with reduced hydrolysis activity and enhanced transglycosylation activity by protein engineering techniques.

[0007] Patent Document 4 describes a method for producing α-anomer-selective glycosides using a glycosyltransferase reaction catalyzed by α-glucosidase from the genus Xanthomonas.

[0008] Patent Document 5 describes that transglycosylation activity is enhanced by introducing a mutation into α-glucosidase derived from Aspergillus niger.

[0009] Non-Patent Document 1 describes that introducing a mutation into α-glucosidase derived from Aspergillus niger allows large amounts of isomaltose and isomaltotriose to accumulate.

[0010] Japanese Patent Application Laid-Open No. 2003-88365 Japanese Patent Application Laid-Open No. 2005-253302 Japanese Patent Application Laid-Open No. 2009-22204 Japanese Patent Application Laid-Open No. 2001-046096 Republished No. 2012-124520

[0011] Min Ma, et al., Applied microbiology and biotechnology, 101(16), 6399-6408, 2017

[0012] However, further improvements in the technology for producing various carbohydrates using transglucosidase are desired.

[0013] Therefore, an object of the present invention is to provide a transglucosidase with modified reaction specificity, and also to provide a method for producing an enzyme preparation, a carbohydrate-containing composition, etc., using the modified transglucosidase.

[0014] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have succeeded in finding a new mutation that can modify the reaction specificity of a transglucosidase derived from an Aspergillus microorganism (Aspergillus niger), thereby completing the present invention. In light of the common technical knowledge that enzymes of the same type have high similarity in structure (primary structure, three-dimensional structure), and that similar mutations are likely to produce similar effects, the mutation technique discovered by the present inventors can be applied not only to the transglucosidase derived from Aspergillus niger shown in the Examples below, but also to transglucosidases derived from other organisms.

[0015] That is, one aspect of the present invention relates to the following: [1] A modified transglucosidase consisting of a polypeptide shown in any one of (1) to (3) below: (1) a polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 1 or 13, into which at least one of the following substitutions has been introduced: (A) a substitution of the amino acid residue at position 227 with a leucine residue or a phenylalanine residue, (B) a substitution of the amino acid residue at position 343 with a tyrosine residue, and (C) a substitution of the amino acid residue at position 493 with a valine residue, (2) a polypeptide into which at least one substitution shown in (A) to (C) has been introduced, into which one or several amino acid residues other than the substituted amino acid residue have been substituted, added, inserted, or deleted, and which has an altered reaction specificity compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 13, (3) A polypeptide having an amino acid sequence into which a substitution shown in at least one of (A) to (C) has been introduced, the sequence identity of the portion excluding the substituted amino acid residues being 70% or more, and having modified reaction specificity relative to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 13. [2] DNA encoding the modified transglucosidase of [1]. [3] An expression cassette or recombinant vector comprising the DNA of [2]. [4] A transformant obtained by introducing the expression cassette or recombinant vector of [3] into a host. [5] A method for producing the modified transglucosidase, comprising the step of culturing the transformant of [4]. [6] An enzyme preparation comprising the modified transglucosidase of [1]. [7] A method for producing a carbohydrate-containing composition, comprising the step of allowing the modified transglucosidase of [1] to act on a carbohydrate-containing raw material. [8] The method of [7], wherein the carbohydrate-containing composition comprises at least one selected from the group consisting of maltotriose, isomaltose, panose, centose, isomaltotriose, 3'-O-α-glucosylmaltose, kojibiose, and nigerose. [9] The method of [7] or [8], wherein the carbohydrate-containing composition is a food or drink.

[0016] The present invention provides a transglucosidase with modified reaction specificity, and also provides an enzyme preparation, a method for producing a carbohydrate-containing composition, and the like, using the modified transglucosidase.

[0017] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited thereto. For convenience of explanation, some of the terms used in connection with the present invention will be defined below.

[0018] (Terminology) As used herein, "transglucosidase" refers to a glucosidase designated by the EC number 3.2.1.20. Transglucosidase is also called α-glucosidase. Transglucosidase is preferably derived from Aspergillus niger. Transglucosidase derived from Aspergillus niger is available, for example, from Amano Enzyme Inc. As used herein, the terms "transglucosidase" and "α-glucosidase" are used interchangeably.

[0019] In this specification, except for the sequence listing, the 20 types of amino acid residues in an amino acid sequence may be represented by single-letter abbreviations: G for glycine (Gly), A for alanine (Ala), V for valine (Val), L for leucine (Leu), I for isoleucine (Ile), F for phenylalanine (Phe), Y for tyrosine (Tyr), W for tryptophan (Trp), S for serine (Ser), T for threonine (Thr), C for cysteine ​​(Cys), M for methionine (Met), D for aspartic acid (Asp), E for glutamic acid (Glu), N for asparagine (Asn), Q for glutamine (Gln), K for lysine (Lys), R for arginine (Arg), H for histidine (His), and P for proline (Pro). In this specification, the amino acid at the mutation introduction point is represented by a combination of a letter representing the type of amino acid and a number representing the position of the amino acid. For example, if the mutation introduction point is tryptophan at position 343, it is represented as "W343."

[0020] In the present specification, the left end of an amino acid sequence is the N-terminus and the right end is the C-terminus.

[0021] As used herein, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0022] As used herein, the term "substitution" refers not only to cases where an amino acid residue substitution is artificially introduced, but also to cases where an amino acid residue substitution is naturally introduced, i.e., cases where the amino acid residue is originally different. As used herein, the amino acid residue substitution may be either an artificial substitution or a natural substitution, with artificial substitution being preferred.

[0023] As used herein, "reaction specificity" refers to the property of producing a specific product when a specific enzyme is allowed to act on a specific substrate. "Modification of reaction specificity" refers to modifying the properties of an enzyme so that it produces different products from the same substrate, and / or modifying the properties of an enzyme so that the production ratio of multiple products produced from the same substrate is changed. The modified transglucosidase of the present invention has modified reaction specificity. The reaction specificity of the modified transglucosidase of the present invention is not particularly limited as long as it is modified compared to the original transglucosidase. For example, the modified transglucosidase of the present invention may have improved reactivity toward α-1,6 bonds (transfer activity and / or degradation activity), reactivity toward α-1,2 bonds (transfer activity and / or degradation activity), or reactivity toward α-1,3 bonds (transfer activity and / or degradation activity). For example, the properties are modified so that the production ratio of at least one of maltotriose, isomaltose, panose, centose, isomaltotriose, 3'-O-α-glucosylmaltose, kojibiose, and nigerose is increased. Preferably, the properties are modified so that the production ratios of maltotriose and centose are increased (mutant 1), the production ratios of maltotriose, isomaltose, centose, kojibiose, and nigerose are increased (mutant 2), the production ratios of maltotriose, centose, 3'-O-glucosylmaltose, kojibiose, and nigerose are increased (mutant 3), the production ratio of panose is increased (mutant 4), or the production ratios of 3'-O-glucosylmaltose, kojibiose, and nigerose are increased (mutant 5).

[0024] Maltotriose, isomaltose, panose, centose, isomaltotriose, 3'-O-α-glucosylmaltose, kojibiose, and nigerose each have the structure shown below. Maltotriose: Glc-α-1,4-Glc-α-1,4-Glc Isomaltose: Glc-α-1,6-Glc Panose: Glc-α-1,6-Glc-α-1,4-Glc Centose: Glc-α-1,2-Glc-α-1,4-Glc Isomaltotriose: Glc-α-1,6-Glc-α-1,6-Glc 3'-O-Glucosylmaltose: Glc-α-1,3-Glc-α-1,4-Glc Kojibiose: Glc-α-1,2-Glc Nigerose: Glc-α-1,3-Glc

[0025] (Modified Transglucosidase) The modified transglucosidase of the present invention comprises a polypeptide shown in any one of (1) to (3) below. (1) A polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 1 or 13, into which at least one of the following substitutions has been introduced: (A) a substitution of the amino acid residue at position 227 with a leucine residue or a phenylalanine residue, (B) a substitution of the amino acid residue at position 343 with a tyrosine residue, or (C) a substitution of the amino acid residue at position 493 with a valine residue; (2) A polypeptide into which at least one of the substitutions shown in (A) to (C) has been introduced, into which one or more amino acid residues other than the substituted amino acid residue have been substituted, added, inserted, or deleted, and which has an altered reaction specificity compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 13; and (3) A polypeptide into which at least one of the substitutions shown in (A) to (C) has been introduced, into which the sequence identity of the portion excluding the substituted amino acid residue is 70% or more, and which has an altered reaction specificity compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 13.

[0026] The polypeptide (1) above is described in more detail below. (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid residue at position 227 has been substituted with a leucine residue or a phenylalanine residue; (b) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid residue at position 343 has been substituted with a tyrosine residue; (c) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid residue at position 493 has been substituted with a valine residue; (d) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid residue at position 227 has been substituted with a leucine residue or a phenylalanine residue, and the amino acid residue at position 343 has been substituted with a tyrosine residue; (e) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid residue at position 227 has been substituted with a leucine residue or a phenylalanine residue, and the amino acid residue at position 493 has been substituted with a valine residue; (f) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid residue at position 343 has been substituted with a tyrosine residue, and the amino acid residue at position 493 has been substituted with a valine residue. (g) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid residue at position 227 is substituted with a leucine residue or a phenylalanine residue, the amino acid residue at position 343 with a tyrosine residue, and the amino acid residue at position 493 with a valine residue; (h) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid residue at position 227 is substituted with a leucine residue or a phenylalanine residue; (i) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid residue at position 343 is substituted with a tyrosine residue; (j) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid residue at position 493 is substituted with a valine residue; (k) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid residue at position 227 is substituted with a leucine residue or a phenylalanine residue, and the amino acid residue at position 343 is substituted with a tyrosine residue.(l) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid residue at position 227 is substituted with a leucine residue or a phenylalanine residue, and the amino acid residue at position 493 is substituted with a valine residue. (m) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid residue at position 343 is substituted with a tyrosine residue, and the amino acid residue at position 493 is substituted with a valine residue. (n) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid residue at position 227 is substituted with a leucine residue or a phenylalanine residue, the amino acid residue at position 343 is substituted with a tyrosine residue, and the amino acid residue at position 493 is substituted with a valine residue.

[0027] In the polypeptide of (2) above, the amino acid modification introduced may include only one type of modification (e.g., substitution only) from substitution, addition, insertion, and deletion, or may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide of (2) above, the number of amino acid differences at any difference site may be one or several, for example, 1 to 80, preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, or 1 to 30, more preferably 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, and particularly preferably 1, 2, or 1.

[0028] Furthermore, in the polypeptide (3), the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 13 may be 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and particularly preferably 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more.

[0029] Here, in the polypeptide (3), the sequence identity to each amino acid sequence shown in SEQ ID NO: 1 or 13 is the sequence identity calculated by comparing with the amino acid sequence shown in SEQ ID NO: 1 or 13. Furthermore, the term "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters are set as follows: Gap insertion cost value: 11, Gap extension cost value: 1.

[0030] In the polypeptides (2) and (3) above, the amino acid residues corresponding to positions 490 (aspartic acid residue) and 660 (aspartic acid residue) in the amino acid sequence shown in SEQ ID NO: 1 or 13 are considered to be active catalytic residues, and therefore it is desirable not to introduce substitutions or deletions at these positions.

[0031] The polypeptides of the present invention may be part of a larger protein (e.g., a fusion protein), in which additional sequences may be added to aid in purification, such as multiple histidine residues, or to ensure stability during recombinant production.

[0032] In the polypeptides of (2) and (3) above, when an amino acid substitution is introduced into SEQ ID NO: 1 or 13, a preferred embodiment of the amino acid substitution to be introduced is a conservative substitution. That is, examples of substitutions in the polypeptides of (2) and (3) above include substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution with another uncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0033] The polypeptides (2) and (3) have α-glucosidase activity and have modified reaction specificity. Modified reaction specificity refers to the property of producing a product with a different composition from that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 13 when reacted with the same substrate. Specifically, this property refers to an increase in the production ratio of panose or centose when maltose is used as a substrate, and the amount of panose or centose produced is 1.1 times or more. Preferably, this is 1.2 times or more, and more preferably, 1.3 times or more. In another embodiment, this property refers to an increase in the production ratio of isomaltose when maltose is used as a substrate. The amount of isomaltose produced is, for example, 1.2 times, preferably 1.5 times or more, or 2 times or more.

[0034]

[0035] Each mutation in the modified transglucosidase of the present invention has advantageous properties compared to the reference enzyme (for example, an enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 or 13), including, but not limited to, the following: G227L: increased α-(1→4) rearrangement and α-(1→2) rearrangement G227F: increased α-(1→4) rearrangement, α-(1→2) rearrangement, and α-(1→3) rearrangement G227F / W343Y: increased α-(1→4) rearrangement, α-(1→2) rearrangement, and α-(1→3) rearrangement, decreased α-(1→6) rearrangement E493V: increased α-(1→6) rearrangement at pH 2.5 W343Y: increased α-(1→2) rearrangement and α-(1→3) rearrangement

[0036] The target enzyme for mutation in the present invention is a transglucosidase. The target enzyme for mutation is typically a wild-type enzyme (an enzyme found in nature). However, the target enzyme may also be an enzyme that has already been mutated or modified in some way. Enzymes to be mutated are not particularly limited, and examples thereof include human maltase-glucoamylase, Aspergillus niger alpha-glucosidase, human neutral alpha-glucosidase C, mouse lysosomal alpha-glucosidase, yeast alpha-glucosidase (Yeast GLU2A), and Aspergillus nidulans alpha-glucosidase A. alpha-glucosidase AgdA), Aspergillus nidulans alpha-glucosidase B (Aspergillus nidulans alpha-glucosidase AgdB), Mucor javanicus alpha-glucosidase (Mucor javanicus alpha-glucosidase), Aspergillus oryzae alpha-glucosidase (Aspergillus oryzae alpha-glucosidase), Mortierella alliacea alpha-glucosidase (Mortierella alliacea alpha-glucosidase), Schizosaccharomyces pombe alpha-glucosidase, Debaryomyces occidentalis alpha-glucosidase, barley alpha-glucosidase (Hordeum vulgare subsp. vulgare alpha-glucosidase), Arabidopsis thaliana alpha-glucosidase,Examples of such transglucosidases include spinach α-glucosidase (Spinacia oleracea alpha-glucosidase), sugar beet α-glucosidase (Beta vulgaris alpha-glucosidase), and potato α-glucosidase (Solanum tuberosum alpha-glucosidase). Preferred is transglucosidase derived from Aspergillus niger.

[0037] (DNA) The DNA of the present invention is a DNA encoding the polypeptide described above. The DNA of the present invention is not particularly limited as long as it has a nucleotide sequence encoding a modified transglucosidase consisting of the polypeptides described above in (1) to (3). The nucleotide sequence shown in SEQ ID NO: 2 is the cDNA sequence of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (sometimes referred to as "Reference Sequence 1"). Therefore, the DNA of the present invention can be appropriately designed by those skilled in the art using SEQ ID NO: 2 as the reference sequence. Furthermore, the nucleotide sequence shown in SEQ ID NO: 14 is the cDNA sequence of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13 (sometimes referred to as "Reference Sequence 2"). Therefore, the DNA of the present invention can be appropriately designed by those skilled in the art using SEQ ID NO: 14 as the reference sequence. Note that position 694 of SEQ ID NO: 1 is alanine, and position 694 of SEQ ID NO: 13 is asparagine.

[0038] Examples of the DNA of the present invention include DNAs shown in any of the following (i) to (iii): (i) DNA consisting of the base sequence shown in SEQ ID NO: 2 or 14, into which at least one of the following substitutions has been introduced: (a) a substitution with a base sequence encoding leucine residues or phenylalanine residues at positions 679 to 681, (b) a substitution with a base sequence encoding tyrosine residues at positions 1027 to 1029, or (c) a substitution with a base sequence encoding valine residues at positions 1477 to 1479, (ii) DNA encoding a polypeptide having an altered reaction specificity for a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or 13, which hybridizes under stringent conditions with DNA having a base sequence complementary to the DNA shown in (i) above, and (iii) DNA encoding a polypeptide having an altered reaction specificity for a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or 13, which has 70% or more homology with the DNA shown in (i) above.

[0039] The DNA of the present invention is not limited to the above sequences, and may have a homology of 70% or more with the base sequences shown in SEQ ID NOs: 8 to 12 and 20 to 24. However, DNA having a base sequence that is preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and particularly preferably 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more homologous thereto is also included in the DNA of the present invention, as long as it encodes a polypeptide having transglucosidase activity.

[0040] Here, DNA "homology" is calculated using publicly available or commercially available software with an algorithm for comparing a reference sequence with a query sequence. Specifically, BLAST, FASTA, or GENETYX (Software Development Co., Ltd.) can be used, and these can be used with default parameters. Note that "homology" can be rephrased as "sequence identity."

[0041] Furthermore, in the base sequences set forth in SEQ ID NOs: 8 to 12 and 20 to 24, mutations such as substitution, addition, insertion or deletion of several bases corresponding to the substitution, addition, insertion or deletion of the amino acid sequences described above are also included in the DNA of the present invention, as long as they encode a polypeptide having transglucosidase activity.

[0042] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequences set forth in SEQ ID NOs: 8 to 12 and 20 to 24 is also included in the DNA of the present invention, as long as it encodes a polypeptide having transglucosidase activity.

[0043] Here, "under stringent conditions" refers to conditions in which the sample is incubated at 50°C to 65°C for 4 hours to overnight in 6xSSC (1xSSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400), and 100 μg / ml salmon sperm DNA.

[0044] Hybridization under stringent conditions is specifically performed as follows. Specifically, a nylon membrane onto which a DNA library or cDNA library is immobilized is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6x SSC, 0.5% SDS, 5x Denhardt's buffer, and 100 μg / ml salmon sperm DNA. Subsequently, each 32P-labeled probe is added, and the membrane is incubated overnight at 65°C. The nylon membrane is washed in 6x SSC at room temperature for 10 minutes, in 2x SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2x SSC containing 0.1% SDS at 45°C for 30 minutes, and then autoradiography is performed to detect DNA that specifically hybridizes with the probe.

[0045] The DNA of the present invention can also be isolated from a microorganism that produces the above-mentioned predetermined polypeptide. For example, the target DNA can be isolated from the genome of Aspergillus niger by PCR or hybridization using the genomic DNA of the microorganism as a template and primers or probes designed from known amino acid sequence information taking into account gene degeneracy, or primers or probes designed based on known nucleotide sequence information.

[0046] The DNA of the present invention encompasses various types of DNA resulting from codon degeneracy. Various types of DNA encoding the same amino acid sequence can be artificially produced easily using known genetic engineering techniques. For example, in the production of a protein by genetic engineering, if the codons used in the original gene encoding the target protein are used infrequently in the host, the expression level of the protein may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon usage frequency for the host without changing the encoded amino acid sequence.

[0047] The sum of the host-optimal codon usage frequencies for each codon can be used as an index of codon usage frequency. An optimal codon is defined as the codon with the highest usage frequency among codons corresponding to the same amino acid. The codon usage frequency is not particularly limited as long as it is optimized for the host. For example, the following is an example of an optimal codon for E. coli: F: phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: histidine (cat), Q: glutamine (cag), N: asparagine (aat), K: lysine (aaa), D: aspartic acid (gat), E: glutamic acid (gaa), S: serine (agc), P: proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine ​​(tgc), W: tryptophan (tgg), R: arginine (cgc), G: glycine (ggc).

[0048] Methods for introducing a mutation into a gene and artificially modifying the amino acid sequence include known techniques such as the Kunkel method and the Gapped duplex method, and mutagenesis kits that utilize site-directed mutagenesis, such as QuikChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), and TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio Inc.).

[0049] The DNA base sequence can be confirmed by conventional sequencing, such as the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be analyzed using an appropriate DNA sequencer.

[0050] Whether the obtained DNA encodes the polypeptide of interest can be confirmed by comparing the determined nucleotide sequence with the nucleotide sequence set forth in SEQ ID NO: 2. Alternatively, the amino acid sequence deduced from the determined nucleotide sequence can be compared with the amino acid sequence set forth in SEQ ID NO: 1 or 13.

[0051] (Expression cassette or recombinant vector) An expression cassette or recombinant vector containing DNA encoding the above-mentioned specified polypeptide (hereinafter also referred to as "expression cassette of the present invention" or "recombinant vector of the present invention") can be obtained by linking a promoter and a terminator to the DNA of the present invention, or by inserting the expression cassette of the present invention or the DNA of the present invention into an expression vector.

[0052] The expression cassette or recombinant vector of the present invention may contain, as control elements, a promoter and a terminator, as well as transcription elements such as an enhancer, a CCAAT box, a TATA box, or an SPI site, as necessary. These control elements may be operably linked to the DNA of the present invention. "Operably linked" means that the DNA of the present invention is linked to various control elements that regulate the DNA of the present invention in a state that allows it to operate in a host cell.

[0053] Regarding the recombinant vector of the present invention, an expression vector constructed for genetic recombination from a phage, plasmid, or virus capable of autonomously replicating in a host is preferred. Such expression vectors are known, and commercially available expression vectors include pQE-based vectors (Qiagen, Inc.), pDR540, pRIT2T (GE Healthcare Biosciences, Inc.), pET-based vectors (Merck & Co., Ltd.), and pUC-based vectors (Takara Bio Inc.). The expression vector may be used in an appropriate combination with the host cell. For example, when Escherichia coli is used as the host cell, examples include a combination of a pET-based vector and a DH5α E. coli strain, a combination of a pET-based vector and a BL21(DE3) E. coli strain, or a combination of a pDR540 vector and a JM109 E. coli strain.

[0054] (Transformant) A transformant (hereinafter also referred to as "the transformant of the present invention") can be obtained by transforming a host with the expression cassette or recombinant vector of the present invention.

[0055] The host used for producing a transformant is not particularly limited, as long as it allows introduction of a gene, the expression cassette or recombinant vector is stable, is capable of autonomous replication, and is capable of expressing the traits of a gene comprising the DNA of the present invention. Suitable examples of the host include bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, and the genus Pseudomonas such as Pseudomonas putida; filamentous fungi such as the genus Aspergillus such as Aspergillus oryzae; and yeast. However, other examples of the host include animal cells, insect cells, and plants.

[0056] The transformant of the present invention can be obtained by introducing the expression cassette of the present invention or the recombinant vector of the present invention into a host. The location of introduction of the DNA of the present invention is not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the expression cassette of the present invention or the recombinant vector of the present invention include, for example, a recombinant vector method and a genome editing method. Conditions for introducing the expression cassette or recombinant vector into a host may be appropriately determined depending on the type of host, etc. When the host is a bacterium, examples include a method using competent cells treated with calcium ions and an electroporation method. When the host is a filamentous fungus, examples include the protoplast PEG method. When the host is a yeast, examples include the electroporation method, the spheroplast method, and the lithium acetate method. When the host is an animal cell, examples include the electroporation method, the calcium phosphate method, and the lipofection method. When the host is an insect cell, examples include the calcium phosphate method, the lipofection method, and the electroporation method. When the host is a plant cell, examples of the method include electroporation, Agrobacterium method, particle gun method, and PEG method.

[0057] Whether or not the expression cassette of the present invention or the recombinant vector of the present invention has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like.

[0058] When confirming by PCR whether the expression cassette of the present invention or the recombinant vector of the present invention has been incorporated into a host, for example, genomic DNA, the expression cassette, or the recombinant vector may be isolated and purified from the transformant.

[0059] For example, when the host is a bacterium, the expression cassette or recombinant vector is isolated and purified using a lysate obtained by lysing the bacterium. Lysis can be achieved by treating the bacterium with a lytic enzyme such as lysozyme, optionally in combination with a protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS).

[0060] Furthermore, physical disruption methods such as freeze-thawing and French press treatment may be combined. DNA can be separated and purified from the lysate by, for example, an appropriate combination of deproteinization treatments using phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and commercially available kits.

[0061] DNA can be cleaved using conventional methods, for example, restriction enzyme treatment. For example, a type II restriction enzyme that acts on a specific nucleotide sequence can be used. DNA can be ligated to an expression cassette or expression vector using, for example, DNA ligase.

[0062] Then, PCR is performed using the isolated and purified DNA as a template and primers specific to the DNA of the present invention. The PCR amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like, and stained with ethidium bromide and SYBR Green solution, etc., and the amplification product is detected as a band, thereby confirming transformation.

[0063] Alternatively, PCR may be performed using primers pre-labeled with a fluorescent dye or the like to detect the amplified product. Furthermore, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and the amplified product is confirmed by fluorescence, enzyme reaction, or the like.

[0064] (Method for Producing the Enzyme) The method for producing the modified transglucosidase of the present invention comprises the step of culturing the transformant of the present invention. Note that, when at least one of the substitutions (A) to (C) contained in the modified transglucosidase is naturally introduced, the modified transglucosidase can be obtained by a production method comprising the step of culturing a microorganism that produces the modified transglucosidase, without transformation using the expression cassette or recombinant vector of the present invention.

[0065] The culture conditions may be appropriately set taking into consideration the nutritional and physiological properties of the transformant or microorganism, but liquid culture is preferred. For industrial production, aeration and agitation culture is preferred. The nutrient source for the medium may be any nutrient required for the growth of the transformant or microorganism. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source may be any assimilable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or alkaline extract of soybean meal. In addition to the carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, as well as specific amino acids and specific vitamins, may also be used as needed.

[0066] The culture temperature can be set appropriately within a range in which the transformant or microorganism of the present invention can grow and the transformant or microorganism can produce the modified transglucosidase, and is preferably about 15 to 37° C. The culture can be completed at an appropriate time when the modified transglucosidase reaches its maximum yield, and can be set appropriately, but the culture time is usually about 12 to 120 hours.

[0067] After culturing the transformant or the microorganism, the culture supernatant or bacterial cells are recovered by centrifugation or other methods, and the bacterial cells are treated with mechanical methods such as ultrasound or a French press, or with a lytic enzyme such as lysozyme, and then solubilized, as necessary, using an enzyme such as protease or a surfactant such as sodium lauryl sulfate (SDS), to obtain a water-soluble fraction containing the modified transglucosidase. That is, in one embodiment of the present invention, the method for producing the modified transglucosidase of the present invention comprises recovering the cultured transformant. In another embodiment of the present invention, the method comprises obtaining the modified transglucosidase from the recovered transformant.

[0068] Furthermore, by selecting an appropriate expression cassette or expression vector and host, the expressed modified transglucosidase can be secreted into the culture medium.

[0069] The water-soluble fraction containing the modified transglucosidase obtained as described above may be subjected to a purification treatment as is, or the modified transglucosidase in the water-soluble fraction may be concentrated and then subjected to a purification treatment.

[0070] The concentration can be carried out by, for example, vacuum concentration, membrane concentration, salting out treatment, fractional precipitation using a hydrophilic organic solvent (for example, methanol, ethanol, and acetone), or the like.

[0071] The above-mentioned predetermined modified transglucosidase can be purified by, for example, an appropriate combination of methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0072] The modified transglucosidase purified in this manner can be powdered by freeze-drying, vacuum drying, spray drying, etc., as needed, and then distributed on the market.

[0073] (Enzyme Preparation) The modified transglucosidase can be provided in the form of an enzyme preparation. Accordingly, the present invention also provides an enzyme preparation containing the modified transglucosidase as an active ingredient.

[0074] The content of the modified transglucosidase in the enzyme preparation of the present invention is not particularly limited, but the lower limit of the content is, for example, 0.01 U / g or more, preferably 0.1 U / g or more, more preferably 1 U / g or more, even more preferably 10 U / g or more, and particularly preferably 100 U / g or more. The upper limit of the content is, for example, 1,000,000 U / g or less, preferably 500,000 U / g or less, 100,000 U / g or less, 50,000 U / g or less, or 10,000 U / g or less.

[0075] The enzyme preparation of the present invention may contain other components in addition to the above-mentioned modified transglucosidase to the extent that the effects of the present invention are not affected. Examples of other components include other enzymes other than the above-mentioned modified transglucosidase, additives, and culture residues generated in the above-mentioned production method.

[0076] Examples of other enzymes include amylases (α-amylase, β-amylase, glucoamylase), glucosidases (β-glucosidase), galactosidases (α-galactosidase, β-galactosidase), proteases (acid proteases, neutral proteases, alkaline proteases), peptidases (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatases (acid phosphatases, alkaline phosphatases), nucleases, deaminases, oxidases, dehydrogenases, glutaminase, pectinases, catalases, dextranases, transglutaminase, protein deamidating enzymes, pullulanases, etc. One of these other enzymes may be contained alone, or multiple types may be contained in combination.

[0077] Examples of additives include excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers include phosphates, citrates, and acetates. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives include ethanol, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, and sodium chloride. These additives may be contained alone or in combination.

[0078] Culture residues include components derived from the culture medium, contaminating proteins, bacterial components, and the like.

[0079] The form of the enzyme preparation of the present invention is not particularly limited, and examples thereof include liquid and solid forms (powder, granules, etc.) The enzyme preparation can be prepared by a generally known method.

[0080] (Use of enzyme) According to the present invention, there is provided a method for producing a carbohydrate-containing composition having an altered sugar composition. Specifically, there are provided methods for producing a carbohydrate-containing composition having an increased panose content, a carbohydrate-containing composition having an increased centose content, a carbohydrate-containing composition having an increased isomaltose content, a carbohydrate-containing composition having an increased maltotriose content, a carbohydrate-containing composition having an increased kojibiose content, a carbohydrate-containing composition having an increased nigerose content, and a carbohydrate-containing composition having an increased 3'-O-α-glucosylmaltose content.

[0081] The method for producing a carbohydrate-containing composition of the present invention is characterized by comprising a step of allowing a modified transglucosidase to act on a carbohydrate-containing raw material. This allows the sugar composition of the resulting carbohydrate-containing composition to be changed. A preferred embodiment is a method for producing a carbohydrate-containing composition having an increased content of maltotriose, isomaltose, panose, centose, isomaltotriose, 3'-O-α-glucosylmaltose, kojibiose, or nigerose, and a more preferred embodiment is a method for producing a carbohydrate-containing composition having an increased content of panose, centose, or isomaltose.

[0082] The carbohydrate-containing raw material used in the present invention is not particularly limited as long as it contains carbohydrates. For example, the type of carbohydrate may be starch, dextrin, oligosaccharide, or maltose. It may contain a single carbohydrate or multiple types of carbohydrates. It may contain only carbohydrates, or may contain components other than carbohydrates (proteins, fats and oils, dietary fiber, inorganic salts, etc.). Examples of carbohydrate-containing raw materials that contain components other than carbohydrates include plant-based raw materials (legumes such as soybeans, peas, lentils, chickpeas, black beans, broad beans, mung beans, lupin beans, and kidney beans; grains such as wheat, barley, oats, rice, rye, buckwheat, barnyard millet, foxtail millet, and teff; nuts such as almonds, coconuts, peanuts, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, pili nuts, chestnuts, sesame seeds, and pine nuts; hemp seeds (industrial hemp), chia seeds, quinoa, amaranth, canary seeds, and flaxseed). The shape of the carbohydrate-containing raw material is not particularly limited. The form may be a solid, liquid or slurry, with liquid or slurry being preferred.

[0083] The amount of modified transglucosidase used is not particularly limited, but may be, for example, 0.0001 mg or more per 1 g of carbohydrate. Preferably, it is 0.001 mg or more, 0.005 mg or more, more preferably 0.01 mg or more, and even more preferably 0.05 mg or more. The upper limit of the amount of modified transglucosidase used per 1 g of carbohydrate is not particularly limited, but may be, for example, 1 g or less, 0.5 g or less, 0.1 g or less, 0.05 g or less, 0.01 g or less, or 0.001 g or less.

[0084] Regarding the hydrolytic activity of transglucosidase, the amount of enzyme that produces 1 μg of glucose in 1 minute using α-methyl-D-glucoside as a substrate is defined as 1 unit (1 U).

[0085] In the present invention, in order to further improve the effect, α-amylase can be used in combination with the above modified transglucosidase. The origin of the α-amylase is not particularly limited, and examples thereof include α-amylases derived from the genus Aspergillus, such as Aspergillus oryzae and Aspergillus niger, and α-amylases derived from the genus Bacillus, such as Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus licheniformis. Preferably, α-amylases derived from the genus Aspergillus are used, and more preferably, α-amylases derived from Aspergillus oryzae are used.

[0086] The amount of α-amylase used is, for example, 0.01 to 100 U, preferably 0.05 to 50 U, more preferably 0.1 to 10 U, and even more preferably 0.3 to 3 U per gram of vegetable protein material.

[0087] Regarding the activity of α-amylase, one unit (1 U) is defined as the amount of enzyme that increases the reducing power equivalent to 10 mg of glucose in 30 minutes using soluble starch as a substrate.

[0088] The reaction time, temperature, and pH of the reaction solution for allowing the modified transglucosidase to act on the carbohydrate-containing raw material are not particularly limited. The reaction temperature is, for example, 10 to 80°C, preferably 20 to 70°C, and more preferably 30 to 60°C. The pH of the reaction solution is, for example, 2 to 9, preferably 3 to 8, and more preferably 3.5 to 7. When the E493V mutant is used, the pH is preferably 2 to 3. The reaction time is, for example, 30 seconds to 48 hours, preferably 1 minute to 24 hours, more preferably 10 minutes to 12 hours, or 30 minutes to 6 hours. When increasing the amount of centose produced, 10 minutes to 1 hour is preferred. When increasing the amount of panose or isomaltose produced, 1 hour to 6 hours is preferred. The sugar composition of the carbohydrate-containing raw material is modified by the above reaction conditions. These reaction conditions are appropriately selected depending on the desired carbohydrate-containing composition. Optimal reaction conditions can be determined through preliminary experiments.

[0089] By using the production method of the present invention, a composition or food with an altered sugar composition can be produced. One embodiment of the production method of the present invention for a carbohydrate-containing composition comprises the following steps (1) and (2). Note that an enzyme deactivation step may be added after step (2): (1) A step of preparing a carbohydrate-containing raw material; (2) A step of treating the prepared raw material with a modified transglucosidase.

[0090] The carbohydrate-containing composition obtained by the manufacturing method of the present invention has an altered sugar composition, and the production ratio of one or more of the carbohydrates panose, centose, isomaltose, and maltotriose is improved compared to carbohydrate-containing compositions obtained by the action of existing transglucosidases (SEQ ID NO: 1 or 13).

[0091] Specific examples of the carbohydrate-containing composition include isomaltooligosaccharide, foods and beverages, and food and beverage ingredients. Examples of foods and beverages include plant-based carbohydrate-containing foods and beverages (such as plant-based milk and plant-based yogurt). Examples of food and beverage ingredients include plant-based food and beverage bases and wort.

[0092] The modified transglucosidase of the present invention can modify the sugar composition of a carbohydrate-containing composition. Therefore, the present invention also provides an agent for modifying the sugar composition of a carbohydrate-containing composition, comprising the modified transglucosidase.

[0093] (1. Preparation of modified enzymes) Using the base sequence (SEQ ID NO: 2) of the transglucosidase gene derived from Aspergillus niger introduced into a pUC119 vector as a template, various mutations were introduced to prepare the following modified transglucosidases. Mutant 1: G227L mutant (SEQ ID NO: 3) of SEQ ID NO: 1 Mutant 2: G227F mutant (SEQ ID NO: 4) of SEQ ID NO: 1 Mutant 3: G227F / W343Y mutant (SEQ ID NO: 5) of SEQ ID NO: 1 Mutant 4: E493V mutant (SEQ ID NO: 6) of SEQ ID NO: 1 Mutant 5: W343Y mutant (SEQ ID NO: 7) of SEQ ID NO: 1

[0094] Specifically, PCR was performed using the following primers and PrimeSTAR (registered trademark) GXL Premix (2x) (manufactured by Takara) in a conventional manner to introduce mutations.

[0095]

[0096] Each mutant gene product was transformed into E. coli JM109 by standard methods to obtain a gene expression vector. The transglucosidase sequence introduced into the gene expression vector was confirmed. The transformants were cultured in LB medium at 37°C for 8 hours with shaking, then subcultured in LB medium and cultured at 37°C for an additional 16 hours with shaking. Bacteria were recovered from the culture, and the plasmid was recovered from the cells using a NucleoBond® Xtra Maxi (manufactured by Takara) according to standard methods. The recovered plasmid was cleaved with the restriction enzyme HindIII, and the desired modified transglucosidase fragment was obtained by gel extraction. The target fragment was then transformed into Aspergillus oryzae using the protoplast-PEG method. The transformants were cultured in a liquid medium containing dextrin, yeast extract, and potassium dihydrogen phosphate at 30°C for 4 days. The culture medium was centrifuged, and the supernatant was collected. After filtering through a 0.45 μm membrane filter, it was desalted and concentrated using an ultrafiltration membrane. Then, it was purified using an anion exchange column to obtain an enzyme sample (Test Examples 1 and 2).

[0097] The modified transglucosidase gene sequence was cloned into pGAPZαA by a standard method, and the prepared plasmid was transformed into Pichia pastoris GS115 by a standard method. The transformant was cultured at 30°C for 48 hours in BMDY medium (20 g / L peptone, 10 g / L yeast extract, 3.4 g / L yeast nitrogen base with amino acids and ammonium sulfate, 10 g / L ammonium sulfate, 0.1 M potassium phosphate (pH 6.0), 10 g / L glucose, and 0.4 mg / L biotin), and then purified using a resin column or the like to obtain an enzyme sample (Test Examples 3 and 4).

[0098] (2. Activity Measurement Method) <Method for Measuring Transglucosidase Hydrolysis Activity> Transglucosidase activity was measured by the following method. (1) 2.0 g of α-methyl-D-glucoside was weighed out and dissolved in water to make 100 mL, to prepare an α-methyl-D-glucoside solution. 1 mL of the α-methyl-D-glucoside solution and 1 mL of 0.02 mol / L acetic acid / sodium acetate buffer (pH 5.0) were weighed into a test tube and left at 40°C for 10 to 15 minutes. Then, 0.5 mL of an aqueous solution containing transglucosidase (enzyme solution) was added and the mixture was shaken well. This was left at 40°C for exactly 60 minutes. After exactly 60 minutes, the mixture was placed in a boiling water bath, heated for exactly 5 minutes, and cooled under running water. (2) 3 mL of the color-developing solution from Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.) was weighed into a test tube, and 0.2 mL of the reaction solution obtained in (1) was added. The mixture was shaken well and then allowed to stand at 40°C for exactly 5 minutes. The absorbance (E60) of this solution at 505 nm was measured using water as a control. Separately, as a blank, 1 mL of 0.02 mol / L acetic acid / sodium acetate buffer (pH 5.0) and 0.5 mL of an aqueous solution containing transglucosidase (enzyme solution) were weighed into a test tube, heated in a boiling water bath for exactly 5 minutes, and then cooled under running water. After cooling, 1 mL of α-methyl-D-glucoside solution was added, and the absorbance (E0) was measured in the same manner as above. (3) Glucose standard solution I or II from Glucose CII-Test Wako was diluted with water to the specified concentration (20 mg / dL or 40 mg / dL). Three mL of Glucose CII Test Wako coloring solution was weighed into a test tube, and 0.2 mL of the above glucose solution was added. The mixture was shaken well and then allowed to stand at 40°C for exactly 5 minutes. The absorbance (ES) of this solution was measured at a wavelength of 505 nm using water as a control. Separately, the absorbance (EB) was measured in the same manner as above using 0.2 mL of water instead of the glucose solution as a blank. A glucose calibration curve was created from the obtained absorbances, and the amount of glucose (μg) (G) at an absorbance difference of 1.000 was calculated. (4) The amount of enzyme required to produce 1 μg of glucose in 60 minutes was defined as 1 unit (1 U), and calculated using the following formula:Transglucosidase activity (U / g, U / mL) = (E60 - E0) x G x 2.5 / 0.2 x n / 0.5 (wherein E60 represents the absorbance of the reaction solution, E0 represents the absorbance of the blank solution, G represents the amount of glucose (µg) when the absorbance difference is 1.000, 2.5 represents the volume of reaction system solution (mL), 0.2 represents the volume of reaction solution collected (mL), 0.5 represents the volume of enzyme solution collected (mL), and n represents the dilution factor per 1 g or 1 mL of sample.)

[0099] (3. Evaluation of Mutants) [Test Example 1] <Evaluation of Transglycosylation Reaction Using Maltose as Substrate-1> Using 3.4% (w / v) maltose / 40 mM sodium acetate buffer (pH 4.0) (Mutant 4 only, 40 mM glycine-HCl (pH 2.5)), the reaction was carried out at 37°C for 5 min, 30 min, 1 hr, 2 hr, and 6 hr using the modified transglucosidase at a final concentration of 0.006%. The resulting sugars (DP1 to DP3) were analyzed by HPLC-RID using a TSKgel Amide-8 0.5 μm 4.6 × 25 cm column. The ratios of the peak area of ​​each sugar to the total peak area were compared. Tables 3 to 5 show the data for the highest yields in the 5-min, 30-min, 1-hr, 2-hr, and 6-hr reactions.

[0100]

[0101]

[0102]

[0103] As shown in Tables 3 to 5, when 3.4% maltose was used as a substrate, Mutants 1 and 2 were shown to have increased maximum maltotriose production compared to the enzyme of Reference Sequence 1. Mutant 3 was shown to have increased maximum maltotriose and centose production compared to the enzyme of Reference Sequence 1. Mutant 4 was shown to have increased maximum panose production compared to the enzyme of Reference Sequence 1.

[0104] Test Example 2: Evaluation of Transglycosylation Reaction Using Maltose as Substrate (2) Using 10% (w / v) maltose / 40 mM sodium acetate buffer (pH 4.0) (Mutant 4 only, 40 mM glycine-HCl (pH 2.5)), the reaction was carried out at 37°C for 5 min, 30 min, 1 hr, 2 hr, and 6 hr using a final concentration of 0.006% modified transglucosidase. The resulting sugars (DP1 to DP3) were analyzed by HPLC-RID using a TSKgel Amide-8 0.5 μm 4.6 × 25 cm column and an MCIGEL COLUMN CK04S column. The ratio of the peak area of ​​each sugar to the total peak area was compared. Tables 6 to 9 show the data for the highest yields in the 5-min, 30-min, 1-hr, 2-hr, and 6-hr reactions.

[0105]

[0106]

[0107]

[0108]

[0109] As shown in Tables 6 to 9, when 10% maltose was used as a substrate, Mutant 1 was shown to have increased maximum production amounts of maltotriose and centose compared to the enzyme of Reference Sequence 1. Mutant 2 was shown to have increased maximum production amounts of isomaltotriose, maltotriose, and centose compared to the enzyme of Reference Sequence 1. Mutant 3 was shown to have increased maximum production amounts of maltotriose and centose compared to the enzyme of Reference Sequence 1. Mutant 4 was shown to have increased maximum production amount of panose compared to the enzyme of Reference Sequence 1.

[0110] [Test Example 3] <Results of Initial Velocity Analysis> 3.4% (w / v) maltose / 40 mM sodium acetate buffer (pH 4.0) (only for Mutant 4, 40 mM glycine-HCl (pH 2.5)) was used as the substrate, and 0.05 U / mL of the modified transglucosidase was reacted at 37°C for 2, 4, 8, 12, and 15 minutes, and the reaction was stopped by maintaining at 100°C for 3 minutes.

[0111] An equal volume of 200 μM sorbitol was added to the reaction stop solution as an internal standard, and 20 μL of the resulting solution was subjected to HPAEC analysis. A CarboPac PA1 Column (Dionex) was used, and elution was performed with 300 mM NaOH (isocratic, 25 minutes). The column temperature was room temperature, and the flow rate was 0.8 mL / min. A calibration curve was prepared using glucose (glc), panose (PN), maltotriose (G3), and centose (2,4-di-O-(α-glucopyranosyl)-glucopyranose, CT) as standard sugars. The results are shown in Table 10.

[0112]

[0113] As shown in Table 10, when 3.4% maltose was used as a substrate, mutants 1, 2, and 3 were shown to have an increased ratio of the initial reaction velocities of maltotriose and centose compared to the enzyme of Reference Sequence 1. Furthermore, mutant 4 was shown to have an increased ratio of the initial reaction velocities of panose compared to the enzyme of Reference Sequence 1.

[0114] Test Example 4 Evaluation of Transglycosylation Reaction Using Maltose as Substrate - 3 3.4% (w / v) maltose / 40 mM sodium acetate buffer (pH 4.0) was used as the substrate, and 0.05 U / mL of the modified transglucosidase was reacted at 37°C for 3 hours, and the reaction was terminated by keeping the temperature at 100°C for 3 minutes.

[0115] The reaction mixture was analyzed by ion chromatography. The results are shown in Tables 11 to 13.

[0116]

[0117]

[0118]

[0119] As shown in Tables 11 to 13, when 3.4% maltose was used as a substrate, Mutant 2 was shown to increase the maximum amounts of kojibiose and nigerose produced compared to the enzyme of Reference Sequence 1. Furthermore, Mutants 3 and 5 were shown to increase the maximum amounts of kojibiose and nigerose produced compared to the enzyme of Reference Sequence 1, and also to produce 3'-O-α-glucosylmaltose, which was not produced by the enzyme of Reference Sequence 1.

[0120] The present invention is extremely useful in various fields using modified transglucosidases due to their modified reaction specificity. This application is based on Japanese Patent Application No. 2024-011342 (filing date: January 29, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A modified transglucosidase consisting of a polypeptide shown in any one of (1) to (3) below: (1) a polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 1 or 13, into which at least one of the following substitutions has been introduced: (A) a substitution of the amino acid residue at position 227 with a leucine residue or a phenylalanine residue, (B) a substitution of the amino acid residue at position 343 with a tyrosine residue, and (C) a substitution of the amino acid residue at position 493 with a valine residue; (2) a polypeptide into which at least one of the substitutions shown in (A) to (C) has been introduced, into which one or more amino acid residues other than the substituted amino acid residue have been substituted, added, inserted, or deleted, and which has an altered reaction specificity compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 13; (3) A polypeptide having an amino acid sequence into which at least one of the substitutions shown in (A) to (C) has been introduced, wherein the sequence identity of the portion excluding the substituted amino acid residues is 70% or more, and the reaction specificity is modified relative to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 13.

2. DNA encoding the modified transglucosidase of claim 1.

3. An expression cassette or recombinant vector containing the DNA of claim 2.

4. A transformant obtained by introducing the expression cassette or recombinant vector according to claim 3 into a host.

5. A method for producing a modified transglucosidase, comprising the step of culturing the transformant according to claim 4.

6. An enzyme preparation comprising the modified transglucosidase of claim 1.

7. A method for producing a carbohydrate-containing composition, comprising the step of reacting the modified transglucosidase of claim 1 with a carbohydrate-containing raw material.

8. The production method according to claim 7, wherein the carbohydrate-containing composition contains at least one selected from the group consisting of maltotriose, isomaltose, panose, centose, isomaltotriose, 3'-O-α-glucosylmaltose, kojibiose, and nigerose.

9. The manufacturing method according to claim 7 or 8, wherein the carbohydrate-containing composition is a food or beverage.

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