Modified transglucosidase

Abstract

Amino acid sequences of the modified transglucosidases, etc. that are constituted by at least any one of substitutions of (A) the 227th amino acid residue to a leucine residue or a phenylalanine residue, (B) the 343rd amino acid residue to a tyrosine residue, and (C) the 493rd amino acid residue to a valine residue introduced in the amino acid sequence shown in SEQ ID NO: 1 or 13 solve the above problems.

Description

Technical Field

[0001] This invention relates to transglucosidases with altered reaction specificity. Furthermore, this invention also relates to the application of such transglucosidases in various uses. Background Technology

[0002] Transglucosidase (α-glucosidase) is primarily an enzyme that hydrolyzes the non-reducing terminal α-1,4-glucosidic bonds of maltodextrins. However, under high substrate concentrations, it can catalyze the formation of isomaltodextrins with α-1,6-glucosidic bonds, exhibiting glycation transfer activity. Transglucosidase is used to manufacture isomaltodextrins from maltose produced by starch breakdown, and is also added during alcoholic beverage production to generate oligosaccharides and improve flavor.

[0003] To date, various reports have been made regarding changes in transglucosidase.

[0004] For example, Patent Document 1 describes how the hydrolytic activity can be reduced to less than one ten-thousandth by introducing a mutation into transglucosidase derived from Saccharomyces cerevisiae.

[0005] Patent document 2 describes that by replacing the aspartic acid or glutamate residue of the nucleophilic catalytic residue of the terminal isomer-retaining glycolytic enzyme with cysteine ​​sulfinic acid residue and / or cysteine ​​sulfonic acid residue, the glycotransfer activity relative to the natural substrate can be improved.

[0006] Patent document 3 describes a method for preparing a protein from glucan glucosidase derived from Streptococcus mutans with reduced hydrolytic activity and enhanced glycan transfer activity through protein engineering.

[0007] Patent document 4 describes a method for manufacturing glycosides that selectively produce α-terminal isomers by utilizing the glycotransfer reaction of α-glucosidase from the genus Xanthomonas.

[0008] Patent document 5 describes how a mutation is introduced into an α-glucosidase derived from Aspergillus niger to make glycemic transfer activity dominant.

[0009] Non-patent literature 1 describes how a large amount of isomaltose and isomalttriose can be accumulated by introducing a mutation into an α-glucosidase derived from Aspergillus niger.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: Japanese Patent Application Publication No. 2003-88365

[0013] Patent Document 2: Japanese Patent Application Publication No. 2005-253302

[0014] Patent Document 3: Japanese Patent Application Publication No. 2009-22204

[0015] Patent Document 4: Japanese Patent Application Publication No. 2001-046096

[0016] Patent Document 5: Publication No. 2012-124520

[0017] Non-patent literature

[0018] Non-patent literature 1: Min Ma, et al., Applied microbiology and biotechnology, 101(16), 6399-6408, 2017 (Min Ma et al., Applied Microbiology and Biotechnology, Vol. 101, No. 16, pp. 6399–6408, 2017) Summary of the Invention

[0019] The problem the invention aims to solve

[0020] However, further technological improvements are expected for the production of various sugars via transglucosidase.

[0021] Therefore, the object of the present invention is to provide a transglucosidase with altered reaction specificity. Furthermore, the object of the present invention is to provide an enzyme preparation using this modified transglucosidase, a method for manufacturing a sugar-containing composition, etc.

[0022] Technical solutions to solve technical problems

[0023] To address the aforementioned challenges, the inventors conducted in-depth research and successfully discovered a novel mutation in transglucosidases derived from *Aspergillus niger* microorganisms that alters reaction specificity, thus completing this invention. Furthermore, given the common technical knowledge that the same mutation produces the same effect due to high structural (primary and stereostructure) similarity, the invention can be applied not only to transglucosidases from *Aspergillus niger* shown in the examples described below, but also to transglucosidases from other biological sources.

[0024] That is, one aspect of the present invention relates to the following.

[0025] [1] A modified transglucosidase, which is a modified transglucosidase composed of any one of the following polypeptides (1) to (3):

[0026] (1) A polypeptide consisting of at least one of the following amino acid sequences introduced into the amino acid sequence shown in sequence number 1 or 13: (A) the amino acid residue at position 227 is replaced by a leucine residue or a phenylalanine residue, (B) the amino acid residue at position 343 is replaced by a tyrosine residue, and (C) the amino acid residue at position 493 is replaced by a valine residue.

[0027] (2) A polypeptide in which one or more amino acid residues other than the substituted amino acid residues are substituted, added, inserted, or deleted in an amino acid sequence containing at least one of the substitutions shown in (A) to (C), and the reaction specificity is altered relative to the polypeptide consisting of the amino acid sequence shown in sequence number 1 or 13, and

[0028] (3) A polypeptide having at least 70% or more sequence identity of the portion of the amino acid sequence excluding the substituted amino acid residues introduced from at least one of (A) to (C) and having a change in reaction specificity relative to the polypeptide consisting of the amino acid sequence shown in sequence number 1 or 13.

[0029] [2] A DNA that encodes the modified transglucosidase described in [1].

[0030] [3] An expression cassette or recombinant vector comprising the DNA described in [2].

[0031] [4] A transformant formed by introducing the expression cassette or recombinant vector described in [3] into a host.

[0032] [5] A method for manufacturing a modified transglucosidase, comprising the step of culturing the transformant described in [4].

[0033] [6] An enzyme preparation comprising the modified transglucosidase described in [1].

[0034] [7] A method for manufacturing a sugar-containing composition, comprising the step of acting the modified transglucosidase described in [1] on a sugar-containing raw material.

[0035] [8] The manufacturing method according to [7], wherein the sugar-containing composition comprises at least one selected from the group consisting of maltotriose, isomaltose, panose, centose, isomaltose, 3'-O-α-glucosylmaltose, kosperidose and nigerose.

[0036] [9] The manufacturing method according to [7] or [8], wherein the sugar-containing composition is a food or beverage.

[0037] The effects of the invention

[0038] According to the present invention, a transglucosidase with altered reaction specificity can be provided. Furthermore, according to the present invention, an enzyme preparation using the modified transglucosidase, a method for manufacturing a sugar-containing composition, etc., can be provided. Detailed Implementation

[0039] The following is a detailed description of one embodiment of the present invention, but the invention is not limited thereto. For ease of explanation, some of the terms used in the present invention are defined below.

[0040] (Terminology)

[0041] In this specification, "transglucosidase" refers to the glucosidase indicated by EC number 3.2.1.20. Transglucosidase is also known as α-glucosidase. Transglucosidase is preferably derived from Aspergillus niger. Aspergillus niger-derived transglucosidase can be obtained, for example, from Amano Enzyme Co., Ltd. In this specification, the terms "transglucosidase" and "α-glucosidase" are used interchangeably.

[0042] In this specification, in addition to the sequence listing, the 20 amino acid residues in the amino acid sequence are sometimes represented by single-letter abbreviations. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine ​​(Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P. In this specification, the amino acid at the mutation introduction point is represented by a combination of a single letter indicating the amino acid type and a number indicating the amino acid position. For example, if the tryptophan at position 343 is the mutation introduction site, it will be represented as "W343".

[0043] In this specification, the amino acid sequence shown has an N-terminus on the left and a C-terminus on the right.

[0044] In this specification, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Non-charged 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.

[0045] In this specification, "substitution" includes not only the case of artificially introduced substitution of amino acid residues, but also the case of naturally introduced substitution of amino acid residues, that is, cases where the original amino acid residues are different. In this specification, the substitution of amino acid residues can be artificial or natural, but artificial substitution is preferred.

[0046] In this specification, "reaction specificity" refers to the property that causes a specific enzyme to produce a specific product when acting on a specific substrate. "Change in reaction specificity" refers to altering the properties of the enzyme to produce different products from the same substrate, and / or altering the properties of the enzyme to change the production ratio of multiple products from the same substrate. The reaction specificity of the modified transglucosidase of the present invention has been altered. The reaction specificity of the modified transglucosidase of the present invention is not particularly limited as long as it has been altered relative to the original transglucosidase. For example, examples include cases where the reactivity (transfer activity and / or degradation activity) of the modified transglucosidase of the present invention to α-1,6 bonds, to α-1,2 bonds, or to α-1,3 bonds is increased. For example, its properties have been altered to increase the production ratio of at least one of maltotriose, isomaltose, panose, santose, isomalttriose, 3'-O-α-glucosylmaltose, kosperidose, and aspergillus niger. Preferably, its properties are altered to increase the production ratio of maltotriose and santose (mutant 1), the production ratio of maltotriose, isomaltose, santose, kosperidose and aspergillus niger (mutant 2), the production ratio of maltotriose, santose, 3'-O-glucosylmaltose, kosperidose and aspergillus niger (mutant 3), the production ratio of panose (mutant 4), and the production ratio of 3'-O-glucosylmaltose, kosperidose and aspergillus niger (mutant 5).

[0047] Maltotriose, isomaltose, panose, santoose, isomaltose, 3'-O-α-glucosylmaltose, kosperidose, and nigerose each have the structures shown below.

[0048] Maltotriose: Glc-α-1,4-Glc-α-1,4-Glc

[0049] Isomaltose: Glc-α-1,6-Glc

[0050] Panose: Glc-α-1,6-Glc-α-1,4-Glc

[0051] Santose: Glc-α-1,2-Glc-α-1,4-Glc

[0052] Isomalttriose: Glc-α-1,6-Glc-α-1,6-Glc

[0053] 3'-O-glucosylmaltose: Glc-α-1,3-Glc-α-1,4-Glc

[0054] Glc-α-1,2-Glc

[0055] Aspergillus niger: Glc-α-1,3-Glc

[0056] (Modified transglucosidase)

[0057] The modified transglucosidase of the present invention is composed of any one of the polypeptides shown in (1) to (3) below.

[0058] (1) A polypeptide consisting of an amino acid sequence formed by replacing the 227th amino acid residue with a leucine or phenylalanine residue, (B) replacing the 343rd amino acid residue with a tyrosine residue, or (C) replacing the 493rd amino acid residue with a valine residue, with at least one of these substitutions introduced into the amino acid sequence shown in sequence number 1 or 13.

[0059] (2) A polypeptide in which one or more amino acid residues other than the substituted amino acid residues are substituted, added, inserted, or deleted in an amino acid sequence shown in at least one of (A) to (C), and the reactivity specificity of the polypeptide relative to the amino acid sequence shown in sequence number 1 or 13 is altered, and

[0060] (3) A polypeptide having at least 70% or more sequence identity with the portion of the substituted amino acid sequence shown in at least one of (A) to (C) and excluding the portion of the substituted amino acid residue, and whose reaction specificity is altered relative to the polypeptide consisting of the amino acid sequence shown in sequence number 1 or 13.

[0061] The polypeptide of (1) is described in more detail below.

[0062] (a) A polypeptide consisting of an amino acid sequence formed by replacing the 227th amino acid residue with a leucine residue or a phenylalanine residue and introducing such a substitution into the amino acid sequence shown in Sequence Number 1.

[0063] (b) A polypeptide consisting of an amino acid sequence formed by replacing the 343rd amino acid residue with a tyrosine residue in the amino acid sequence shown in Sequence Number 1.

[0064] (c) A polypeptide consisting of an amino acid sequence formed by replacing the 493rd amino acid residue with a valine residue in the amino acid sequence shown in Sequence Number 1.

[0065] (d) A polypeptide consisting of an amino acid sequence formed by replacing the 227th amino acid residue with a leucine or phenylalanine residue and replacing the 343rd amino acid residue with a tyrosine residue, which is introduced into the amino acid sequence shown in Sequence Number 1.

[0066] (e) A polypeptide consisting of an amino acid sequence formed by replacing the 227th amino acid residue with a leucine or phenylalanine residue and replacing the 493rd amino acid residue with a valine residue, which is introduced into the amino acid sequence shown in Sequence Number 1.

[0067] (f) A polypeptide consisting of an amino acid sequence formed by replacing the 343rd amino acid residue with a tyrosine residue and the 493rd amino acid residue with a valine residue in the amino acid sequence shown in Sequence Number 1.

[0068] (g) A polypeptide consisting of an amino acid sequence formed by replacing the 227th amino acid residue with a leucine or phenylalanine residue, replacing the 343rd amino acid residue with a tyrosine residue, and replacing the 493rd amino acid residue with a valine residue in the amino acid sequence shown in Sequence Number 1.

[0069] (h) A polypeptide consisting of an amino acid sequence in which the amino acid residue at position 227 is replaced by a leucine residue or a phenylalanine residue, and this substitution is introduced into the amino acid sequence shown in sequence number 13.

[0070] (i) A polypeptide consisting of an amino acid sequence formed by replacing the 343rd amino acid residue with a tyrosine residue and introducing that substitution into the amino acid sequence shown in sequence number 13.

[0071] (j) A polypeptide consisting of an amino acid sequence formed by replacing the 493rd amino acid residue with a valine residue and introducing that substitution into the amino acid sequence shown in sequence number 13.

[0072] (k) A polypeptide consisting of an amino acid sequence formed by replacing the 227th amino acid residue with a leucine or phenylalanine residue and replacing the 343rd amino acid residue with a tyrosine residue, which is introduced into the amino acid sequence shown in sequence number 13.

[0073] (l) A polypeptide consisting of an amino acid sequence formed by replacing the 227th amino acid residue with a leucine or phenylalanine residue and replacing the 493rd amino acid residue with a valine residue, which is introduced into the amino acid sequence shown in sequence number 13.

[0074] (m) A polypeptide consisting of an amino acid sequence formed by replacing the 343rd amino acid residue with a tyrosine residue and the 493rd amino acid residue with a valine residue, which is introduced into the amino acid sequence shown in sequence number 13.

[0075] (n) A polypeptide consisting of an amino acid sequence formed by replacing the 227th amino acid residue with a leucine or phenylalanine residue, the 343rd amino acid residue with a tyrosine residue, and the 493rd amino acid residue with a valine residue, in the amino acid sequence shown in sequence number 13.

[0076] In the polypeptide of (2), the modified amino acid can be a single modification including substitution, addition, insertion, and deletion (e.g., substitution only), or it can include two or more modifications (e.g., substitution and insertion). In the polypeptide of (2), the number of different amino acids at any different site can be one or more, 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, further preferably 1 to 3, and particularly preferably 1 or 2, or 1.

[0077] In addition, in the polypeptide of (3), the sequence identity with respect to the amino acid sequence shown in sequence number 1 or 13 is only 70% or more, preferably 75% or more, more preferably 80% or more, further 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, and 99% or more.

[0078] Here, in the polypeptide of (3), the sequence identity relative to each amino acid sequence shown in sequence number 1 or 13 is the sequence identity calculated by comparing it with the amino acid sequence shown in sequence number 1 or 13. Additionally, "Sequence Identity" refers to the identity value of amino acid sequences obtained through the bl2seq program in the BLASTPACKAGE (SGI 32-bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)). The parameters can be set to 11 for gap insertion cost and 1 for gap extension cost.

[0079] In the polypeptides of (2) and (3), the amino acid residues at positions 490 (aspartic acid residue) and 660 (aspartic acid residue) of the amino acid sequence corresponding to sequence number 1 or 13 are considered to be active catalytic residues, and therefore it is desirable not to introduce substitutions or deletions at these sites.

[0080] The polypeptide of the present invention can be part of a larger protein (e.g., a fusion protein). Examples of sequences added to the fusion protein include sequences that facilitate purification, such as those with multiple recombinant residues, and sequences that ensure stability during recombinant production.

[0081] In the polypeptides of (2) and (3), when an amino acid substitution is introduced relative to sequence number 1 or 13, a suitable manner of introducing the amino acid substitution can be listed as a conservative substitution. That is, as a substitution in the polypeptides of (2) and (3), for example, if the amino acid before substitution is a nonpolar amino acid, it is substituted with another nonpolar amino acid; if the amino acid before substitution is an uncharged amino acid, it is substituted with another uncharged amino acid; if the amino acid before substitution is an acidic amino acid, it is substituted with another acidic amino acid; and if the amino acid before substitution is a basic amino acid, it is substituted with another basic amino acid.

[0082] The polypeptides in (2) and (3) possess α-glucosidase activity, and their reaction specificity is altered. The altered reaction specificity refers to the characteristic that, when used with the same substrate, it produces a product with a composition different from the polypeptide composed of the amino acid sequence shown in sequence number 1 or 13. Specifically, it is the characteristic that the production rate of panose or santose increases when maltose is used as a substrate, and the amount of panose or santose produced is 1.1 times or more. Preferably, it is 1.2 times or more, more preferably 1.3 times or more. Alternatively, it is the characteristic that the production rate of isomaltose increases 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.

[0083] Table 1

[0084]

[0085] The mutations in the modified transglucosidases of the present invention, compared to the reference enzyme (e.g., an enzyme consisting of the amino acid sequence shown in sequence number 1 or 13), possess, for example, the following advantageous properties, but are not limited thereto.

[0086] G227L: Increased α-(1→4) and α-(1→2) transfers

[0087] G227F: Increased α-(1→4) and α-(1→2) and α-(1→3) transfers

[0088] G227F / W343Y: Increased α-(1→4) and α-(1→2) and α-(1→3) transfers, and decreased α-(1→6) transfers.

[0089] E493V: Increased α-(1→6) transfer at pH 2.5

[0090] W343Y: Increased α-(1→2) and α-(1→3) transfers

[0091] The mutant target enzyme in this invention is transglucosidase. The mutant target enzyme is a typical wild-type enzyme (an enzyme found in nature). However, an enzyme that has already undergone some mutation or modification can also be used as the mutant target enzyme. While there are no specific limitations on the enzymes that can be considered as targets for mutation, examples include: human maltase-glucoamylase, Aspergillus niger alpha-glucosidase, human neutral alpha-glucosidase C, mouse lysosomal alpha-glucosidase, yeast GLU2A, Aspergillus nidulans alpha-glucosidase AgdA, Aspergillus nidulans alpha-glucosidase AgdB, and Mucorjavanicus alpha-glucosidase. Alpha-glucosidase, alpha-glucosidase from Aspergillus oryzae, alpha-glucosidase from Mortierella alliacea, alpha-glucosidase from Schizosaccharomyces pombe, alpha-glucosidase from Debaryomyces occidentalis, alpha-glucosidase from barley (Hordeum vulgare subsp. vulgare), alpha-glucosidase from Arabidopsis thaliana, alpha-glucosidase from spinach (Spinacia oleracea), and alpha-glucosidase from beets (Beta vulgaris) Transglucosidase, such as alpha-glucosidase from Solanum tuberosum, is preferred.

[0092] (DNA)

[0093] The DNA of the present invention is DNA encoding the polypeptide described above. The DNA of the present invention is not particularly limited to any DNA having a base sequence encoding a modified transglucosidase composed of the polypeptides shown in (1) to (3) described above. The base sequence shown in Sequence Number 2 is a cDNA sequence encoding a polypeptide composed of the amino acid sequence shown in Sequence Number 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 Sequence Number 2 as a reference sequence. Furthermore, the base sequence shown in Sequence Number 14 is a cDNA sequence encoding a polypeptide composed of the amino acid sequence shown in Sequence Number 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 Sequence Number 14 as a reference sequence. Additionally, position 694 of Sequence Number 1 is alanine, and position 694 of Sequence Number 13 is asparagine.

[0094] Examples of DNA in this invention include any one of the DNAs shown in (i) to (iii) below.

[0095] DNA consisting of at least one of the following substitutions: (a) substitution to a base sequence encoding a leucine residue or a phenylalanine residue at positions 679–681; (b) substitution to a base sequence encoding a tyrosine residue at positions 1027–1029; or (c) substitution to a base sequence encoding a valine residue at positions 1477–1479, introduced into the base sequence shown in sequence number 2 or 14.

[0096] (ii) DNA encoding a polypeptide whose reaction specificity has been altered relative to the amino acid sequence shown in sequence number 1 or 13, and DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA shown in (i) above, and

[0097] (iii) DNA encoding a polypeptide whose reaction specificity has been altered relative to the amino acid sequence represented by sequence number 1 or 13, and which is DNA with more than 70% homology to the DNA represented by (i).

[0098] The DNA of the present invention is not limited to the above-described sequences. It is acceptable as long as the DNA has 70% or more homology with the base sequences shown in sequence numbers 8-12 and 20-24. However, it is preferred to have 75% or more, more preferably 80% or more, further preferably 85% or more, and even more preferably 90% or more. DNA with base sequences having particularly preferred homology of 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, as long as it encodes a polypeptide with transglucosidase activity, is also included in the DNA of the present invention.

[0099] Here, DNA "homology" is calculated using publicly available or commercially available software with an algorithm that compares a reference sequence as a query sequence. Specifically, software such as BLAST, FASTA, or GENETYX (manufactured by Software Development Corporation) can be used with these set to default parameters. Furthermore, "homology" can be translated as "sequence identity".

[0100] In addition, corresponding to the aforementioned substitutions, additions, insertions, or deletions of amino acid sequences, mutated base sequences in the base sequences described in sequence numbers 8-12 and 20-24 that have undergone substitutions, additions, insertions, or deletions of several bases are also included in the DNA of the present invention, as long as they encode a polypeptide with transglucosidase activity.

[0101] Furthermore, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA sequenced in sequence numbers 8-12 and 20-24, as long as it encodes a polypeptide with transglucosidase activity, is also included in the DNA of the present invention.

[0102] Here, "strict conditions" refers to incubation at 50°C to 65°C for 4 hours to overnight in 6×SSC (1×SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5× Denhardt's [Denhardt's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% polysaccharide 400] and 100 μg / ml salmon sperm DNA.

[0103] Hybridization under stringent conditions is specifically performed using the following method: A nylon membrane immobilized with a DNA or cDNA library is prepared and blocked at 65°C in a prehybridization solution containing 6×SSC, 0.5% SDS, 5×Denhardt's, and 100 μg / ml salmon sperm DNA. Subsequently, probes labeled with 32P are added, and the membrane is incubated overnight at 65°C. The membrane is then washed in 6×SSC at room temperature for 10 minutes, in 2×SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2×SSC containing 0.1% SDS at 45°C for 30 minutes. Autoradiography is then performed to detect DNA that has specifically hybridized with the probes.

[0104] The DNA of the present invention can also be isolated from microorganisms that produce the aforementioned polypeptides. For example, using the genomic DNA of Aspergillus niger as a template, the target DNA can be isolated from the genome of the microorganism by using PCR or hybridization with primers or probes designed based on known amino acid sequence information and taking into account gene degeneracy, or primers or probes designed based on known base sequence information.

[0105] The DNA of this invention comprises multiple degenerate DNAs derived from codons. Multiple DNAs encoding the same amino acid sequence can be easily created artificially using known genetic engineering techniques. For example, in the production of genetically engineered proteins, protein expression levels are sometimes low when the codons used on the original gene encoding the target protein are codons with low usage frequency in the host. In such cases, high expression of the target protein can be achieved by optimizing the codon usage frequency in the host without modifying the encoded amino acid sequence.

[0106] As an indicator of codon usage frequency, the sum of the host-optimized codon usage frequencies for each codon is sufficient. An optimal codon is defined as the codon with the highest usage frequency among codons corresponding to the same amino acid. There is no particular limitation on codon usage frequency as long as it is optimized for the host, but examples of optimal codons in *E. coli* can be listed below. 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 (tggg), R: Arginine (cgc), G: Glycine (ggc).

[0107] As a method for artificially modifying amino acid sequences by introducing mutations into genes, known techniques such as the Kunkel method or the Gapped duplex method can be used, as well as mutation introduction kits that utilize site-specific mutagenesis, such as the 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).

[0108] The determination of the DNA base sequence can be performed using conventional methods. For example, it can be done using dideoxynucleotide chain termination (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be resolved using a suitable DNA sequencer.

[0109] To confirm whether the obtained DNA is the DNA encoding the target polypeptide, it can be done by comparing the determined base sequence with the base sequence recorded in Sequence Number 2. Alternatively, it can be done by comparing the amino acid sequence deduced from the determined base sequence with the amino acid sequence recorded in Sequence Number 1 or 13.

[0110] (Expression cassette or recombinant vector)

[0111] Expression cassettes or recombinant vectors containing DNA encoding the aforementioned polypeptides (hereinafter also referred to as "expression cassettes of the present invention" or "recombinant vectors 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.

[0112] In the expression cassette or recombinant vector of the present invention, in addition to promoters and terminators, transcriptional elements such as enhancers, CCAAT boxes, TATA boxes, and SPI sites may be further included as needed as control factors. These control factors only need to be functionally linked to the DNA of the present invention. Functionally linked means that the various control factors regulating the DNA of the present invention and the DNA of the present invention are linked in a state where they can function in the host cell.

[0113] Regarding the recombinant vector of the present invention, as an expression vector, it is preferably a vector constructed from a bacteriophage, plasmid, or virus capable of autonomous replication within a host for gene recombination. Such expression vectors are well known; for example, commercially available expression vectors include pQE line vectors (Qiagen), pDR540, pRIT2T (GE Healthcare Biosciences), pET line vectors (Merck), and pUC line vectors (Takara Bio Inc.). The expression vector can be used simply by selecting an appropriate combination with the host cell. For example, when using *E. coli* as the host cell, combinations such as pET line vector and DH5α *E. coli* strain, pET line vector and BL21(DE3) *E. coli* strain, or pDR540 vector and JM109 *E. coli* strain can be used.

[0114] (Transformation)

[0115] By using the expression cassette or recombinant vector of the present invention to transform the host, a transformant (hereinafter also referred to as "the transformant of the present invention") can be obtained.

[0116] As a host used in the manufacture of the transformant, there are no particular limitations as long as the gene can be introduced, the expression cassette or recombinant vector is stable, and it can autonomously proliferate to express the trait of the gene containing the DNA of the present invention. For example, bacteria belonging to the genera such as Escherichia coli, Bacillus genus such as Bacillus subtilis, and Pseudomonas genus such as Pseudomonas putida; filamentous fungi such as Aspergillus oryzae; and yeast are listed as preferred examples. In addition, animal cells, insect cells, plants, etc. can also be used.

[0117] The transformants of the present invention can be obtained by introducing the expression cassette or recombinant vector of the present invention into a host. The location where the DNA of the present invention is introduced is not particularly limited, as long as the target gene can be expressed; it can be located on a plasmid or on the genome. Specific methods for introducing the expression cassette or recombinant vector of the present invention include, for example, the recombinant vector method and the genome editing method. The conditions for introducing the expression cassette or recombinant vector into the host can be appropriately set according to the type of host, etc. When the host is bacteria, methods using competent cells treated with calcium ions, electroporation, etc., are examples. When the host is filamentous fungi, methods such as the protoplast PEG method are examples. When the host is yeast, methods such as electroporation (electroporation), protoplasts, and lithium acetate are examples. When the host is animal cells, methods such as electroporation, calcium phosphate, and liposome transfection are examples. When the host is insect cells, methods such as calcium phosphate, liposome transfection, and electroporation are examples. When the host is plant cells, methods such as electroporation, Agrobacterium, gene gun, and PEG are examples.

[0118] Confirmation of whether the expression cassette or recombinant vector of the present invention has been integrated into the host can be performed by PCR, Southern hybridization, and Northern hybridization, etc.

[0119] When it is confirmed by PCR whether the expression cassette or recombinant vector of the present invention has been integrated into the host, for example, it is sufficient to isolate and purify the genomic DNA or expression cassette or recombinant vector from the transformant.

[0120] The isolation and purification of expression cassettes or recombinant vectors, for example in the case of bacteria, are based on the lysates obtained by lysing the bacteria. Methods of lysis include treatment with lysins such as lysozymes, and, if necessary, with the addition of proteases and other enzymes, as well as surfactants such as sodium dodecyl sulfate (SDS).

[0121] Furthermore, physical disruption methods such as freeze-thaw cycles and French press can be combined. DNA can be isolated and purified from the lysate, for example, by appropriately combining protein removal processes such as phenol and protease treatment, ribonuclease treatment, alcohol precipitation, and commercially available kits.

[0122] DNA cutting can be performed using conventional methods, such as restriction enzyme treatment. For example, type II restriction enzymes that act on specific nucleotide sequences can be used. The binding of DNA to an expression cassette or expression vector can be performed, for example, using DNA ligase.

[0123] Subsequently, using the isolated and purified DNA as a template, primers specific to the DNA of this invention were designed for PCR. The amplification products obtained by PCR were subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide and SYBR Green solution, etc. The amplification products were detected as bands, thereby confirming that transformation had been achieved.

[0124] Alternatively, PCR can be performed beforehand using primers labeled with fluorescent dyes, and the amplification products can be detected. Furthermore, methods can be employed to bind the amplification products to a solid phase such as a microplate and confirm the amplification products through fluorescence and enzyme reactions.

[0125] (Enzyme manufacturing methods)

[0126] The method for manufacturing the modified transglucosidase of the present invention includes a step of culturing the transformant of the present invention. Furthermore, when at least one of the substitutions represented by (A) to (C) in the modified transglucosidase is naturally introduced, the modified transglucosidase can be obtained by a manufacturing method including a step of culturing microorganisms that produce the modified transglucosidase, without using the expression cassette or recombinant vector of the present invention for transformation.

[0127] The culture conditions can be appropriately set considering the nutritional and physiological properties of the transformant or the microorganism, but liquid culture is preferred. Furthermore, in the case of industrial manufacturing, aerated and stirred culture is preferred. As the nutrient source for the culture medium, substances required for the growth of the transformant or the microorganism can be used. As a carbon source, any commercially available carbon compound can be used, such as glucose, sucrose, lactose, maltose, molasses, pyruvate, etc. As a nitrogen source, any commercially available nitrogen compound can be used, such as peptone, meat extract, yeast extract, casein hydrolysate, soybean meal alkali extract, etc. In addition to carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins can be used as needed.

[0128] The culture temperature can be appropriately set within a range that allows the transformant or microorganism of the present invention to grow and produce modified transglucosidase, but is preferably around 15 to 37°C. The culture should be completed at an appropriate time, as long as the estimated period for the modified transglucosidase to reach its maximum yield is reached. This can be appropriately set, and the culture time is typically around 12 to 120 hours.

[0129] After culturing the transformant or the microorganism, the culture supernatant or bacterial cells are recovered from the culture medium by methods such as centrifugation. The bacterial cells are then treated by mechanical methods such as ultrasound and Freund's crushing, or by lysing enzymes such as lysozyme. If necessary, enzymes such as proteases or surfactants such as sodium dodecyl sulfate (SDS) are used to solubleize the cells, thereby obtaining a water-soluble fraction containing the modified transglucosidase. That is, in one embodiment of the present invention, the method for manufacturing the modified transglucosidase includes a step of recovering the cultured transformant. Furthermore, in another embodiment of the present invention, a step of obtaining the modified transglucosidase from the recovered transformant is included.

[0130] In addition, by selecting appropriate expression cassettes or expression vectors and hosts, it is also possible to secrete the expressed modified transglucosidase into the culture medium.

[0131] The water-soluble fraction containing the modified transglucosidase obtained as described above can be used directly for purification, or it can be used for purification after concentrating the modified transglucosidase in the water-soluble fraction.

[0132] Concentration can be achieved through methods such as vacuum concentration, membrane concentration, salting out, or fractional precipitation using hydrophilic organic solvents (e.g., methanol, ethanol, and acetone).

[0133] The purification process of the specified modified transglucosidase can be carried out by appropriate combination of methods such as gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.

[0134] The purified modified transglucosidase can be powdered as needed through freeze drying, vacuum drying, spray drying, etc., so that it can be sold on the market.

[0135] (Enzyme)

[0136] Modified transglucosidases can be provided in the form of enzyme preparations. Therefore, the present invention also provides enzyme preparations containing modified transglucosidases as active ingredients.

[0137] The content of the modified transglucosidase in the enzyme agent of the present invention is not particularly limited, but as a lower limit, examples include 0.01 U / g or more, preferably 0.1 U / g or more, more preferably 1 U / g or more, further preferably 10 U / g or more, and particularly preferably 100 U / g or more. As an upper limit, examples include 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, and 10,000 U / g or less.

[0138] In addition to the specified modified transglucosidase, the enzyme preparation of the present invention may also contain other components to a degree that does not affect the effects of the present invention. Examples of other components include enzymes other than the specified modified transglucosidase, additives, and culture residues generated during the manufacturing method.

[0139] Other enzymes include, but are not limited to, amylases (α-amylase, β-amylase, glucosylamylase), glucosidases (β-glucosidase), galactosidases (α-galactosidase, β-galactosidase), proteases (acidic proteases, neutral proteases, alkaline proteases), peptidases (leucine peptidase, aminopeptidase), lipases, esterases, cellulases, phosphatases (acid phosphatase, alkaline phosphatase), nucleases, deaminases, oxidases, dehydrogenases, glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deaminases, pullulanase, etc. These other enzymes can be present individually or in combinations of multiple enzymes.

[0140] Examples of additives include excipients, buffers, suspending agents, stabilizers, preservatives, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, and glycerin. 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, p-hydroxybenzoic acid, chlorobutanol, and sodium chloride. These additives can be present individually or in combination.

[0141] As culture residues, the following can be listed: components from the culture medium source, miscellaneous proteins, bacterial cell components, etc.

[0142] The form of the enzyme agent of the present invention is not particularly limited, and examples include liquid, solid form (powder, granules, etc.). The enzyme agent can be prepared by generally known methods.

[0143] (Uses of enzymes)

[0144] According to the present invention, a method for manufacturing a sugar-containing composition in which the sugar composition has been altered is provided. Specifically, methods for manufacturing sugar-containing compositions with increased panose content, sugar-containing compositions with increased sansose content, sugar-containing compositions with increased isomaltose content, sugar-containing compositions with increased maltotriose content, sugar-containing compositions with increased kosherb content, sugar-containing compositions with increased aspergillose content, and sugar-containing compositions with increased 3'-O-α-glucosylmaltose content are provided.

[0145] The method for manufacturing the sugar-containing composition of the present invention is characterized by including a step of acting a modified transglucosidase on the sugar-containing raw material. This allows for a change in the sugar composition of the obtained sugar-containing composition. Preferably, the method is a method for manufacturing a sugar-containing composition with increased content of maltotriose, isomaltose, panose, santelose, isomaltose, 3'-O-α-glucosylmaltose, kosperidose, or aspergillus niger. More preferably, the method is a method for manufacturing a sugar-containing composition with increased content of panose, santelose, or isomaltose.

[0146] The sugar-containing raw materials used in this invention are not particularly limited as long as they contain sugar. For example, the type of sugar can be starch, dextrin, oligosaccharides, or maltose. It can be a single sugar or contain multiple sugars. It can contain only sugar or contain other components (proteins, fats, dietary fiber, inorganic salts, etc.). Examples of sugar-containing raw materials that do not contain sugar include plant-based raw materials (soybeans, green peas, lentils, chickpeas, black beans, broad beans, mung beans, lupins, kidney beans, etc.); cereals such as wheat, barley, oats, rice, rye, buckwheat, barnyard millet, millet, teff, etc.; nuts such as almonds, coconuts, peanuts, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, prickly ash, chestnuts, sesame seeds, pine nuts, etc.; and chia seeds, quinoa, amaranth, canary grass seeds, flax seeds, etc.). The form of the sugar-containing raw material is not particularly limited. Solid, liquid, and paste forms are acceptable, with liquid and paste forms being preferred.

[0147] There is no particular limitation on the amount of modified transglucosidase used, but examples of amounts per 1g of sugar include 0.0001 mg or more. Preferably, amounts of 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. There is no particular upper limit to the range of amounts of modified transglucosidase used per 1g of sugar, and examples of amounts less than 1g, less than 0.5g, less than 0.1g, less than 0.05g, less than 0.01g, or less than 0.001g are possible.

[0148] Regarding the hydrolytic activity of transglucosidase, using α-methyl-D-glucosidase as a substrate, the amount of enzyme that generates 1 μg of glucose per minute is defined as 1 unit (1U).

[0149] In this invention, to further improve the effect, α-amylase can be used in conjunction with the modified transglucosidase. The source of the α-amylase is not particularly limited, but examples include α-amylases from the genus *Aspergillus*, such as *Aspergillus oryzae* and *Aspergillus niger*, and α-amylases from the genus *Bacillus*, such as *Bacillus amyloliquefaciens*, *Bacillus subtilis*, and *Bacillus licheniformis*. Preferably, α-amylases from the genus *Aspergillus* are used, and more preferably, α-amylases from the genus *Aspergillus oryzae* are used.

[0150] Regarding the amount of α-amylase used, for each 1g of plant protein raw material, examples include 0.01 to 100U, preferably 0.05 to 50U, more preferably 0.1 to 10U, and even more preferably 0.3 to 3U.

[0151] Regarding the activity of α-amylase, using soluble starch as a substrate, the amount of enzyme that brings an increase in reducing power equivalent to 10 mg of glucose within 30 minutes is defined as 1 unit (1U).

[0152] The reaction time, temperature, and pH of the reaction solution for inducing the modified transglucosidase to act on the sugar-containing raw material are not particularly limited. The reaction temperature is, for example, 10–80°C, preferably 20–70°C, more preferably 30–60°C. The pH of the reaction solution is, for example, 2–9, preferably 3–8, more preferably 3.5–7. When using the E493V mutant, the pH is preferably 2–3. The reaction time is, for example, 30 seconds–48 hours, preferably 1 minute–24 hours, more preferably 10 minutes–12 hours or 30 minutes–6 hours. When it is desired to increase the production of santose, 10 minutes–1 hour is preferred. When it is desired to increase the production of panose or isomaltoose, 1 hour–6 hours is preferred. The above reaction conditions aim to change the sugar composition of the sugar-containing raw material. These reaction conditions are appropriately selected according to the desired sugar-containing composition. Furthermore, the optimal reaction conditions can be determined through preliminary experiments.

[0153] By using the manufacturing method of the present invention, it is possible to manufacture compositions or foods with altered sugar composition. One embodiment of the manufacturing method of the sugar-containing composition of the present invention includes the following steps (1) and (2). Furthermore, an enzyme inactivation step may be added after step (2).

[0154] (1) Process of preparing sugary raw materials

[0155] (2) The process of processing the prepared raw materials using modified transglucosidase.

[0156] The sugar composition of the sugar-containing composition obtained by the manufacturing method of the present invention is changed, and the production ratio of any one or more sugars among panose, sandeose, isomaltose, and maltotriose is increased compared with the sugar-containing composition obtained by acting an existing transglucosidase (sequence number 1 or 13).

[0157] Specific forms of sugar-containing compositions include isomaltooligosaccharides, food products, or food product ingredients. Food products include those containing plant-based sugars (plant-based milk, plant-based yogurt, etc.). Food product ingredients include plant-based food bases or wort.

[0158] The modified transglucosidase of the present invention can alter the sugar composition of a sugar-containing composition. Therefore, the present invention also provides a sugar composition modifier comprising a modified transglucosidase in a sugar-containing composition.

[0159] Example

[0160] (1. Preparation of modified enzymes)

[0161] Using the base sequence (sequence number 2) of the transglucosidase gene from Aspergillus niger introduced into the pUC119 vector as a template, various mutations were introduced to produce the following modified transglucosidase.

[0162] Mutant 1: G227L mutant of sequence number 1 (sequence number 3)

[0163] Mutant 2: G227F mutant of sequence number 1 (sequence number 4)

[0164] Mutant 3: The G227F / W343Y mutant of sequence number 1 (sequence number 5)

[0165] Mutant 4: E493V mutant of sequence number 1 (sequence number 6)

[0166] Mutant 5: The W343Y mutant of sequence number 1 (sequence number 7)

[0167] Specifically, the mutation was introduced by PCR using the following primers and PrimeSTAR (registered trademark) GXL Premix (2×) (manufactured by Takara) via conventional methods.

[0168] Table 2

[0169]

[0170] The obtained mutant gene products were transformed into *E. coli* JM109 using conventional methods to obtain gene expression vectors. The transglucosidase sequence introduced into the gene expression vectors was confirmed. Transformants were cultured in LB medium at 37°C with shaking for 8 hours, then subcultured into LB medium and cultured for another 16 hours with shaking at 37°C. Cells were recovered from the culture, and plasmids were recovered from the cells using a NucleoBond (registered trademark) Xtra Maxi (manufactured by Takara) according to conventional methods. The recovered plasmids were cleaved with the restriction enzyme HindIII, and the target modified transglucosidase fragment was obtained by gel extraction. This fragment was then transformed into *Aspergillus oryzae* using the protoplast-PEG method. Transformants were cultured in 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 recovered. After filtering through a 0.45 μm filter membrane, the supernatant was desalted and concentrated using an ultrafiltration membrane. Subsequently, the enzyme samples were purified by anion exchange column and used as enzyme samples (Examples 1 and 2).

[0171] The modified transglucosidase gene sequence was cloned into pGAPZαA using conventional methods. The prepared transglucosidase was then transformed into Pichia pastoris GS115 using conventional methods. The transformants were cultured at 30°C for 48 h in BMDY medium (20 g / L peptone, 10 g / L yeast extract, 3.4 g / L yeast nitrogen base without amino acids and ammonium sulfate, 10 g / L ammonium sulfate, 0.1 M potassium phosphate buffer (pH 6.0), 10 g / L glucose, and 0.4 mg / L biotin). After purification by resin column chromatography, the enzyme samples were obtained (Examples 3 and 4).

[0172] (2. Activity assay method)

[0173] <Assay for transglucosidase hydrolysis activity>

[0174] The activity of transglucosidase was determined by the following method.

[0175] (1) Weigh 2.0 g of α-methyl-D-glucosinolate and dissolve it in water to a final volume of 100 mL to prepare an α-methyl-D-glucosinolate solution. Weigh 1 mL of the α-methyl-D-glucosinolate solution and 1 mL of 0.02 mol / L acetate-sodium acetate buffer (pH 5.0) into a test tube. After standing at 40 °C for 10–15 minutes, add 0.5 mL of an aqueous solution containing transglucosidase (enzyme solution) and shake thoroughly to mix. Place the solution at 40 °C for exactly 60 minutes. After exactly 60 minutes, place the solution in a boiling water bath and heat for exactly 5 minutes, then cool it under running water.

[0176] (2) Weigh 3 mL of glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) colorimetric solution into a test tube, add 0.2 mL of the reaction solution obtained in (1), shake thoroughly to mix, and incubate at 40°C for 5 minutes. Using water as a control, measure the absorbance (E60) at a wavelength of 505 nm. As a blank, weigh 1 mL of 0.02 mol / L acetate-sodium acetate buffer (pH 5.0) and 0.5 mL of an aqueous solution containing transglucosidase (enzyme solution) into a test tube, heat accurately in a boiling water bath for 5 minutes, and then cool under running water. After cooling, add 1 mL of α-methyl-D-glucosidase solution, perform the same operation as described above, and measure the absorbance (E0).

[0177] (3) Dilute glucose standard solution I or II of glucose CII-Test Wako with water to the specified concentration (20 mg / dL, 40 mg / dL). Weigh 3 mL of glucose CII-Test Wako colorimetric solution into a test tube, add 0.2 mL of the above glucose solution, shake thoroughly to mix, and incubate accurately at 40°C for 5 minutes. Measure the absorbance (ES) at a wavelength of 505 nm using water as a control. Alternatively, perform the same operation using 0.2 mL of water instead of the glucose solution as a blank, and measure the absorbance (EB). Plot a standard curve of glucose from the obtained absorbance values, and determine the amount of glucose (μg) (G) when the absorbance difference is 1.000.

[0178] (4) The amount of enzyme that generates 1 μg of glucose within 60 minutes is taken as 1 unit (1U) and calculated by the following formula.

[0179] Transglucosidase activity (U / g, U / mL) = (E60 - E0) × G × 2.5 / 0.2 × n / 0.5

[0180] (In the formula, 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 the reaction system solution (mL), 0.2 represents the volume of the reaction solution collected (mL), 0.5 represents the volume of the enzyme solution collected (mL), and n represents the dilution factor per 1g or 1mL of sample.)

[0181] (3. Evaluation of mutants)

[0182] [Experimental Example 1]

[0183] <Evaluation of maltose-based glycan transfer reactions - 1>

[0184] Using 3.4% (w / v) maltose / 40 mM sodium acetate buffer (pH 4.0) (mutant 4 only, 40 mM glycine-HCl (pH 2.5)) as substrate, modified transglucosidase was used at a final concentration of 0.006% at 37 °C for 5 min, 30 min, 1 hr, 2 hr, and 6 hr. The generated sugars (DP1–DP3) were analyzed by HPLC-RID using a TSKgel Amide-80.5 μm 4.6 × 25 cm column. The peak area of ​​each sugar was compared to the total peak area. The data for the highest yield at 5 min, 30 min, 1 hr, 2 hr, and 6 hr are shown in Tables 3–5.

[0185] Table 3

[0186]

[0187] Table 4

[0188]

[0189] Table 5

[0190]

[0191] As shown in Tables 3-5, when using 3.4% maltose as a substrate, mutants 1 and 2 showed an increased maximum production of maltotriose compared to the enzyme of reference sequence 1. Additionally, mutant 3 showed an increased maximum production of both maltotriose and santose compared to the enzyme of reference sequence 1. Furthermore, mutant 4 showed an increased maximum production of panose compared to the enzyme of reference sequence 1.

[0192] [Experimental Example 2]

[0193] <Evaluation of maltose-based glycan transfer reactions - 2>

[0194] Using 10% (w / v) maltose / 40 mM sodium acetate buffer (pH 4.0) (mutant 4 only, 40 mM glycine-HCl (pH 2.5)) as substrate, modified transglucosidase was used at a final concentration of 0.006% at 37 °C for 5 min, 30 min, 1 hr, 2 hr, and 6 hr. The generated sugars (DP1–DP3) were analyzed by HPLC-RID using a TSKgel Amide-80.5 μm 4.6 × 25 cm column and an MCIGEL COLUMN CK04S column. The peak area of ​​each sugar was compared to the total peak area. The data for the highest yield at 5 min, 30 min, 1 hr, 2 hr, and 6 hr are shown in Tables 6–9.

[0195] Table 6

[0196]

[0197] Table 7

[0198]

[0199] Table 8

[0200]

[0201] Table 9

[0202]

[0203] As shown in Tables 6-9, when using 10% maltose as a substrate, mutant 1 exhibits increased maximum production of maltotriose and Santose compared to the enzyme of reference sequence 1. Furthermore, mutant 2 shows increased maximum production of isomaltoriose, maltotriose, and Santose compared to the enzyme of reference sequence 1. Further, mutant 3 shows increased maximum production of maltotriose and Santose compared to the enzyme of reference sequence 1. Additionally, mutant 4 shows increased maximum production of panose compared to the enzyme of reference sequence 1.

[0204] [Experimental Example 3]

[0205] <Analytical results of initial velocity>

[0206] Using 3.4% (w / v) maltose / 40 mM sodium acetate buffer (pH 4.0) (mutant 4 only, 40 mM glycine-HCl (pH 2.5)) as substrate, the modified transglucosidase at 0.05 U / mL was reacted at 37 °C for 2, 4, 8, 12, and 15 minutes, and the reaction was terminated by holding at 100 °C for 3 minutes.

[0207] An equal volume of 200 μM sorbitol was added to the reaction termination solution as an internal standard, and 20 μL of this standard was analyzed by HPAEC. A CarboPac PA1 column (manufactured by Dionex) was used, and elution was performed with 300 mM NaOH (isocratic elution, 25 min). The column temperature was set to room temperature, and the flow rate was set to 0.8 mL / min. A standard curve was prepared using glucose (glc), panose (PN), maltotriose (G3), and santelose (2,4-di-O-(α-glucopyranosyl)-glucopyranose, CT) as standard sugars. The results are shown in Table 10.

[0208] Table 10

[0209]

[0210] Table 10 shows the increased ratio of initial reaction rates of maltotriose and santose in mutants 1, 2, and 3 compared to the enzyme of baseline sequence 1 when using 3.4% maltose as a substrate. Additionally, it shows the increased ratio of initial reaction rates of panose in mutant 4 compared to the enzyme of baseline sequence 1.

[0211] [Experimental Example 4]

[0212] <Evaluation of maltose-based glycan transfer reactions - 3>

[0213] Using 3.4% (w / v) maltose / 40 mM sodium acetate buffer (pH 4.0) as a substrate, 0.05 U / mL of the modified transglucosidase was reacted at 37 °C for 3 hours, and the reaction was terminated by holding at 100 °C for 3 minutes.

[0214] The reaction termination solution was analyzed by ion chromatography. The results are shown in Tables 11–13.

[0215] Table 11

[0216]

[0217] Table 12

[0218]

[0219] Table 13

[0220]

[0221] As shown in Tables 11-13, when using 3.4% maltose as a substrate, mutant 2 showed an increased maximum production of kosperidin and aspergillus niger compared to the enzyme of reference sequence 1. Furthermore, mutants 3 and 5, compared to the enzyme of reference sequence 1, not only showed an increased maximum production of kosperidin and aspergillus niger, but also produced 3'-O-α-glucosylmaltose, which was not produced in the enzyme of reference sequence 1.

[0222] Industrial utilization potential

[0223] This invention is extremely useful in various fields that utilize modified transglucosidases due to the altered reaction specificity. This application is based on Japanese Patent Application No. 2024-011342 (filed January 29, 2024), the contents of which are fully incorporated herein by reference.

Claims

1. A modified transglucosidase, which is a modified transglucosidase composed of any one of the following polypeptides (1) to (3): (1) A polypeptide consisting of at least one of the following amino acid sequences introduced into the amino acid sequence shown in sequence number 1 or 13: (A) the amino acid residue at position 227 is replaced by a leucine residue or a phenylalanine residue, (B) the amino acid residue at position 343 is replaced by a tyrosine residue, and (C) the amino acid residue at position 493 is replaced by a valine residue. (2) A polypeptide in which one or more amino acid residues other than the substituted amino acid residues are substituted, added, inserted, or deleted in an amino acid sequence containing at least one of the substitutions shown in (A) to (C), and the reaction specificity is altered relative to the polypeptide consisting of the amino acid sequence shown in sequence number 1 or 13, and (3) A polypeptide having at least 70% or more sequence identity of the portion of the amino acid sequence containing at least one of the substituted amino acid residues shown in (A) to (C) and having a change in reaction specificity relative to the polypeptide consisting of the amino acid sequence shown in sequence number 1 or 13.

2. A DNA that encodes the modified transglucosidase of claim 1.

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

4. A transformant formed by introducing the expression cassette or recombinant vector of claim 3 into a host.

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

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

7. A method for manufacturing a sugar-containing composition, comprising the step of acting the modified transglucosidase of claim 1 on a sugar-containing raw material.

8. The manufacturing method according to claim 7, wherein, The sugar-containing composition comprises at least one sugar selected from the group consisting of maltotriose, isomaltose, panose, santoose, isomaltose, 3'-O-α-glucosylmaltose, kosperidose, and aspergillus niger.

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

Images