glucosyltransferase

Abstract

Provided is a novel glucosyltransferase. The present technology provides a glucosyltransferase which includes a polypeptide mentioned in any one of the following (1) to (4). (1) A polypeptide which comprises an amino acid sequence represented by SEQ ID NO: 1 or 2; (2) a polypeptide which has a structure such that one or several amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 or 2 are substituted, added, inserted, or deleted, and which has a glucosyltransferase activity; (3) a polypeptide which has 93% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and which has a glucosyltransferase activity; and (4) a polypeptide which has 90% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and which has a glucosyltransferase activity.

Description

Glucosyltransferase

[0001] This technology relates to glucosyltransferase. More specifically, this technology relates to a novel glucosyltransferase, DNA encoding the glucosyltransferase, an expression cassette or recombinant vector containing the DNA, a transformant, a method for producing glucosyltransferase, a method for producing one or more saccharides selected from α-1,3 glucan and leucrose, a method for producing food and drink products, a method for reducing saccharides in food and drink products, food and drink products, and an enzyme agent for reducing saccharides.

[0002] Glucosyltransferase is an enzyme that forms glycosidic bonds and catalyzes reactions that hydrolyze substrates and transfer sugars. A typical target substrate for glucosyltransferase is sucrose. Since such catalytic ability can be applied to various chemical reactions, it is expected to be used in the food industry as a sugar-reducing enzyme.

[0003] For example, Patent Document 1 discloses that by contacting water, sucrose, and a glucosyltransferase enzyme that synthesizes poly-α-1,3-glucan having at least 30% α-1,3-linkages to perform an enzymatic reaction, an aqueous composition containing at least 55% fructose on a dry weight basis and containing an insoluble poly-α-1,3-glucan product is produced.

[0004] Also, for example, Patent Document 2 discloses a technique for producing insoluble α-1,3-glucan by contacting at least water, sucrose, a glucosyltransferase enzyme, and (i) an oligosaccharide containing α-1,3 and α-1,6 glycosidic linkages, or (ii) an oligosaccharide produced from a glucosyltransferase reaction.

[0005] Further, for example, Patent Document 3 discloses a method for reducing the monosaccharide and / or disaccharide content in a food material by contacting a glucosyltransferase containing a specific amino acid sequence with the food material.

[0006] International Publication No. 2017 / 106262, International Publication No. 2018 / 217722, International Publication No. 2016 / 173929

[0007] As mentioned above, glucosyltransferases are expected to have widespread applications in industry. These applications include catalyzing reactions that reduce sugars (monosaccharides and disaccharides) in food and produce oligosaccharides (trisaccharides or more) and polysaccharides. However, currently, glucosyltransferases produced and put into practical use on an industrial scale are not being used for sugar reduction purposes.

[0008] Therefore, the primary objective of this technology is to provide a novel glucosyltransferase.

[0009] This technology first provides a glucosyltransferase comprising a polypeptide as shown in any of the following (1) to (4): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (2) a polypeptide having glucosyltransferase activity, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 or 2 are substituted, added, inserted, deleted, or inverted; (3) a polypeptide having glucosyltransferase activity, wherein the amino acid sequence shown in SEQ ID NO: 1 has a sequence identity of 93% or more to the amino acid sequence shown in SEQ ID NO: 1; and (4) a polypeptide having glucosyltransferase activity, wherein the amino acid sequence shown in SEQ ID NO: 2 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 2. The glucosyltransferase according to this technology may be derived from a microorganism of the genus Streptococcus. Furthermore, the glucosyltransferase according to this technology may be derived from Streptococcus thermophilus.

[0010] This technology then provides DNA comprising any of the following polynucleotides: (1) a polynucleotide encoding the glucosyltransferase described in claim 1; (2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 or 6; (3) a polynucleotide encoding a polypeptide having glucosyltransferase activity and comprising a sequence equivalent to the base sequence shown in SEQ ID NO: 5 or 6. This technology also provides an expression cassette or recombinant vector comprising the DNA relating to this technology. This technology also provides a transformant obtained by transforming a host with the expression cassette or recombinant vector relating to this technology.

[0011] This technology also provides a method for producing glucosyltransferase, comprising: a culture step of culturing a microorganism of the genus Streptococcus or a transformant related to this technology; and a recovery step of recovering glucosyltransferase related to this technology from the culture obtained in the culture step.

[0012] This technology further provides a method for producing one or more carbohydrates selected from α-1,3-glucan and leucose, comprising the step of reacting sucrose with the glucosyltransferase described in claim 1. This technology also provides a method for producing food and beverages, comprising the step of reacting sugar-containing food and beverages and / or their raw materials with a glucosyltransferase containing a polypeptide shown in any of (1) to (4) above. In addition, this technology provides a method for reducing sugars in food and beverages, comprising the step of reacting sugar-containing food and beverages and / or their raw materials with a glucosyltransferase containing a polypeptide shown in any of (1) to (4) above. This technology also provides food and beverages obtained using the glucosyltransferase according to this technology. This technology also provides an enzyme preparation for sugar reduction, comprising the glucosyltransferase according to this technology.

[0013] The following is an explanation of the technical terms used in this technology. <Amino Acid Notation> The 20 amino acid residues in an amino acid sequence are sometimes represented by a single-letter abbreviation. In other words, glycine (Gly) is sometimes represented by G, alanine (Ala) by A, valine (Val) by V, leucine (Leu) by L, isoleucine (Ile) by I, phenylalanine (Phe) by F, tyrosine (Tyr) by Y, tryptophan (Trp) by W, serine (Ser) by S, threonine (Thr) by T, cysteine ​​(Cys) by C, methionine (Met) by M, aspartic acid (Asp) by D, glutamic acid (Glu) by E, asparagine (Asn) by N, glutamine (Gln) by Q, lysine (Lys) by K, arginine (Arg) by R, histidine (His) by H, and proline (Pro) by P.

[0014] <Amino Acid Sequence> In this technology, the displayed amino acid sequence has the N-terminus on the left and the C-terminus on the right.

[0015] <Types of Amino Acids> In this technology, "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.

[0016] <Sequence Modification (Substitution, Addition, Insertion, Deletion, Inversion, etc.)> In this technology, modification of amino acid sequences and / or base sequences includes not only artificially performed modifications (substitution, addition, insertion, deletion, inversion, etc.) but also naturally occurring modifications (substitution, addition, insertion, deletion, inversion, etc.), i.e., cases where the sequences were originally different.

[0017] <Isolation and Purification> In this technology, the term "isolated" is used interchangeably with "purified." The term "isolated" is used to distinguish it from its natural state, that is, the state in which it exists in nature. The artificial operation of "isolation" results in an "isolated state," which is a state different from the natural state. The isolated substance is clearly and definitively different from the natural product itself.

[0018] <Equivalent Sequences> In this technology, an "equivalent sequence" refers to an amino acid sequence or nucleotide sequence that differs in part from a reference amino acid sequence (SEQ ID NO: 1 or SEQ ID NO: 2 in this technology) or nucleotide sequence (SEQ ID NO: 5 or SEQ ID NO: 6 in this technology), but whose differences do not substantially affect the function of the protein (glucosyltransferase activity in this technology). Therefore, in this technology, enzymes having a polypeptide chain consisting of an equivalent amino acid sequence, or enzymes encoded by polynucleotides consisting of an equivalent nucleotide sequence, exhibit glucosyltransferase activity.

[0019] <Sugars> In this technology, "sugars" refers to the general term for monosaccharides and disaccharides (excluding sugar alcohols) as defined in the Nutrition Labeling Standards (Ministry of Health, Labour and Welfare Notification No. 176). Monosaccharides are sugars that cannot be broken down further, and include glucose, fructose, and galactose. Disaccharides include sucrose (glucose + fructose), maltose (glucose + glucose), lactose (glucose + galactose), etc.

[0020] This graph shows the optimal temperature for either enzyme 1 (a glucosyltransferase containing a polypeptide with the amino acid sequence shown in SEQ ID NO: 1) or enzyme 2 (a glucosyltransferase containing a polypeptide with the amino acid sequence shown in SEQ ID NO: 2).

[0021] The following describes preferred embodiments for implementing this technology. Note that the embodiments described below are merely examples of typical embodiments of this technology, and this should not be interpreted as narrowing the scope of this technology.

[0022] 1. Glucosyltransferase The glucosyltransferase related to this technology will be described in detail below.

[0023] [Amino Acid Sequence] The glucosyltransferase related to this technology comprises a polypeptide as shown in any of the following (1) to (4): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (2) a polypeptide having glucosyltransferase activity, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 or 2 are substituted, added, inserted, deleted, or inverted; (3) a polypeptide having glucosyltransferase activity, wherein the amino acid sequence shown in SEQ ID NO: 1 has a sequence identity of 93% or more to the amino acid sequence shown in SEQ ID NO: 1; and (4) a polypeptide having glucosyltransferase activity, wherein the amino acid sequence shown in SEQ ID NO: 2 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 2.

[0024] In the polypeptide of (2) above, the modification of amino acids may include only one type of modification (e.g., only substitution) from among substitution, addition, insertion, deletion, and inversion, or it 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 or 2 or 1.

[0025] In the polypeptide of (3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 should be 93% or more, but preferably 94% or more, more preferably 95% or more, even more preferably 96% or more, and even more preferably 97% or more, 98% or more, or 99% or more.

[0026] In the polypeptide of (4) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 2 should be 90% or more, but preferably 93% or more, more preferably 95% or more, even more preferably 96% or more, and even more preferably 97% or more, 98% or more, or 99% or more.

[0027] Here, in the polypeptides of (3) and (4) above, the sequence identity with respect to each amino acid sequence shown in SEQ ID NO: 1 or 2 is the sequence identity calculated by comparing it with the amino acid sequence shown in SEQ ID NO: 1 or 2. Furthermore, "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 National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0028] Furthermore, it is desirable not to introduce substitutions or deletions to the active site in the polypeptides described in (2) to (4) above.

[0029] The polypeptide of this technology may be part of a larger protein (e.g., a fusion protein). Examples of sequences that can be added to a fusion protein include sequences useful for purification, such as multiple histidine residues, and sequences that ensure stability during recombinant production.

[0030] In the polypeptides of (2) to (4) above, when an amino acid substitution is introduced for SEQ ID NO: 1 or 2, a preferred embodiment of the introduced amino acid substitution is a conservative substitution. Specifically, substitutions in the polypeptides of (2) to (4) above include, for example, substitution of another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution of another noncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution of another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution of another basic amino acid if the amino acid before substitution is a basic amino acid.

[0031] It is preferable that the glucosyltransferase containing the polypeptides (2) to (4) has glucosyltransferase activity equal to or greater than that of the glucosyltransferase containing the polypeptide (1). Specifically, the relative activity of the glucosyltransferase containing the polypeptides (2) to (4) is 0.7 times or more, 0.8 times or more, 0.9 times or more, 1.0 times or more, 1.1 times or more, preferably 1.15 times or more, more preferably 1.2 times or more, and even more preferably 1.25 times or more, than the relative activity of the glucosyltransferase containing the polypeptide (1).

[0032] [Origin] The glucosyltransferase related to this technology is not particularly limited in its origin, as long as it contains the polypeptide described above. For example, glucosyltransferases derived from bacteria (e.g., Streptococcus genus) and filamentous fungi (e.g., Aspergillus genus) are examples. Preferably, glucosyltransferases derived from microorganisms of the genus Streptococcus are examples, and more preferably, glucosyltransferases derived from Streptococcus thermophilus are examples.

[0033] Here, "glucosyltransferase derived from Streptococcus thermophilus" refers to glucosyltransferase produced by microorganisms classified as Streptococcus thermophilus (whether wild-type or mutant), or glucosyltransferase obtained through genetic engineering using the glucosyltransferase gene. Therefore, recombinant organisms produced by host microorganisms into which the glucosyltransferase gene obtained from Streptococcus thermophilus (or a modified version of that gene) has been introduced also fall under the category of "glucosyltransferase derived from Streptococcus thermophilus."

[0034] The strain of Streptococcus thermophilus is not particularly limited as long as it does not impair the action or effect of this technology, and strains that are generally available from biological genetic resource institutions, etc., can be used.

[0035] More specifically, the glucosyltransferase containing the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is a glucosyltransferase derived from Streptococcus thermophilus. The amino acid sequence, including the signal sequence and pro sequence, is shown in SEQ ID NO: 3.

[0036] The glucosyltransferase containing the polypeptide with the amino acid sequence shown in SEQ ID NO: 2 is a glucosyltransferase derived from Streptococcus thermophilus. The amino acid sequence, including the signal sequence and pro sequence, is shown in SEQ ID NO: 4.

[0037] Furthermore, the glucosyltransferase related to this technology may be an artificial protein artificially designed based on the amino acid sequences described in (1) to (4) above.

[0038] [Enzymatic Chemical Properties] (1) Action The glucosyltransferase related to this technology is an enzyme that hydrolyzes sucrose to produce one or more carbohydrates selected from α-1,3-glucan and leucrose. The α-1,3-glucan that the glucosyltransferase related to this technology can produce includes insoluble α-1,3-glucan and soluble α-1,3-glucan. Furthermore, the insoluble α-1,3-glucan that the glucosyltransferase related to this technology can produce includes seven or more sugars. The soluble α-1,3-glucan that the glucosyltransferase related to this technology can produce includes nigerooligosaccharides such as nigerose, nigerotriose, nigerotetraose, nigeropentaose, and nigerohexaose. In addition, the glucosyltransferase related to this technology may also be an enzyme that has other actions, as long as it has glucosyltransferase activity.

[0039] (2) Optimal temperature The optimal temperature for the glucosyltransferase related to this technology is 30°C. The optimal temperature is the value obtained when using 50 mM potassium phosphate buffer pH 7.0.

[0040] (3) Molecular weight The molecular weight of the glucosyltransferase related to this technology is 168 kDa. The molecular weight was measured using SDS-PAGE.

[0041] 2. DNA encoding glucosyltransferase The DNA relating to this technology includes one of the following polynucleotides: (1) a polynucleotide encoding the glucosyltransferase relating to this technology; (2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 or 6; (3) a polynucleotide encoding a polypeptide having glucosyltransferase activity, which includes a sequence equivalent to the base sequence shown in SEQ ID NO: 5 or 6.

[0042] The "polynucleotide encoding the glucosyltransferase according to the present technology" in the above (1) refers to a polynucleotide that can obtain the glucosyltransferase according to the present technology when it is expressed. It includes not only a polynucleotide having a base sequence corresponding to the amino acid sequence of the glucosyltransferase according to the present technology, but also a polynucleotide obtained by adding a sequence that does not encode an amino acid sequence to such a polynucleotide. It also includes a polynucleotide with degenerate codons.

[0043] Specific examples of the polynucleotide encoding the glucosyltransferase according to the present technology are shown in SEQ ID NO: 5 (genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 1), SEQ ID NO: 7 (cDNA sequence encoding the amino acid sequence of SEQ ID NO: 3), SEQ ID NO: 6 (genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 2), and SEQ ID NO: 8 (cDNA sequence encoding the amino acid sequence of SEQ ID NO: 4).

[0044] The DNA according to the present technology includes a polynucleotide containing a sequence (hereinafter also referred to as an "equivalent sequence") that has a different base sequence in part although the function of the protein it encodes is equivalent when compared with the base sequence of the polynucleotide encoding the glucosyltransferase according to the present technology in the above (1).

[0045] Specific examples of the equivalent sequence include a base sequence consisting of a modified base sequence including substitution, addition, insertion, deletion, or inversion of one or more bases based on the base sequence of the polynucleotide encoding the glucosyltransferase according to the present technology (for example, SEQ ID NO: 5, 6, 7, or 8), and encoding a polypeptide having glucosyltransferase activity.

[0046] The base modification may occur at multiple sites in the sequence. Here, "multiple" varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the DNA, but is, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases.

[0047] Further, as specific examples of equivalent sequences, with respect to the base sequence of the polynucleotide encoding the glucosyltransferase according to the present technology (for example, SEQ ID NO: 5, 6, 7, or 8), sequences having a sequence identity of, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, still more preferably about 90% or more, even still more preferably 95% or more, and particularly preferably 99% or more can be mentioned.

[0048] Here, "sequence identity" can be calculated using publicly available or commercially available software having an algorithm for comparing a reference sequence as a query sequence. Specifically, BLAST, FASTA, or GENETYX (manufactured by Software Development Co., Ltd.) etc. can be used, and these may be used with the default parameters set.

[0049] In the present technology, DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 5, 6, 7, or 8, and DNA that hybridizes under stringent conditions are also included in the DNA according to the present technology as long as this encodes a polypeptide having glucosyltransferase activity.

[0050] Here, "under stringent conditions" refers to conditions of incubating 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% ficoll 400] and 100 μg / ml salmon sperm DNA.

[0051] Hybridization under stringent conditions is specifically performed by the following method: A nylon membrane immobilized with a DNA library or cDNA library is prepared, and the nylon membrane is blocked at 65°C in a pre-hybridization solution containing 6×SSC, 0.5% SDS, 5×Denharts, and 100 μg / mL salmon sperm DNA. Then, each probe labeled with 32P is added, and the membrane is incubated overnight at 65°C. After washing the nylon membrane 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 can be performed to detect DNA that has specifically hybridized with the probe.

[0052] [Method for obtaining DNA related to this technology] The DNA related to this technology can be prepared in an isolated state by standard genetic engineering methods, molecular biological methods, biochemical methods, chemical synthesis, PCR (e.g., overlap PCR), or a combination thereof, with reference to the sequence information disclosed in this specification or the attached sequence listing.

[0053] Furthermore, the DNA related to this technology can also be isolated from microorganisms that produce polypeptides having glucosyltransferase activity. For example, the target DNA can be isolated from the genome of the microorganism by PCR or hybridization using primers or probes designed considering gene degeneracy from known amino acid sequence information, or primers or probes designed based on known base sequence information.

[0054] The DNA related to this technology encompasses various types of DNA derived from codon degeneracy. Artificially creating multiple types of DNA encoding the same amino acid sequence can be easily done using known genetic engineering techniques. For example, in the production of genetically engineered proteins, if the codons used in the original gene encoding the target protein are infrequently used in the host, the protein expression level may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon utilization frequency for the host without altering the encoded amino acid sequence.

[0055] As an indicator of codon utilization frequency, the sum of the host-optimal codon utilization frequencies for each codon can be adopted. An optimal codon is defined as the codon with the highest utilization frequency among codons corresponding to the same amino acid. Codon utilization frequency is not particularly limited as long as it is optimized for the host, but the following are examples of optimal codons in 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).

[0056] Methods for introducing mutations into genes and artificially modifying amino acid sequences include known methods such as the Kunkel method and the Gapped duplex method, as well as mutation introduction kits utilizing site-directed mutagenesis, such as the QuickChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), and the TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio).

[0057] The base sequence of DNA can be confirmed by sequencing using conventional methods. For example, it can be done by dideoxynucleotide chain termination (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be analyzed using a suitable DNA sequencer.

[0058] To confirm whether the obtained DNA encodes the target polypeptide, the determined base sequence can be compared with the base sequence described in SEQ ID NO: 7 or 8. Alternatively, the amino acid sequence predicted from the determined base sequence can be compared with the amino acid sequence described in SEQ ID NO: 1 or 2.

[0059] [Uses of DNA related to this technology] The DNA related to this technology can be used as a probe for identifying the gene encoding the glucosyltransferase related to this technology, the DNA encoding the glucosyltransferase related to this technology, or as a primer for amplifying or mutating the DNA encoding the glucosyltransferase related to this technology.

[0060] The DNA related to this technology can be used to prepare the glucosyltransferase related to this technology. A genetic engineering preparation method using the DNA related to this technology makes it possible to obtain a more homogeneous glucosyltransferase related to this technology. Furthermore, this method is also suitable for preparing large quantities of the glucosyltransferase related to this technology. If the DNA related to this technology is a polynucleotide DNA that does not contain a start codon, the glucosyltransferase related to this technology can be obtained by adding a start codon or a signal peptide containing a start codon and then expressing it.

[0061] The DNA related to this technology can also be used as an experimental tool for elucidating the mechanism of action of the glucosyltransferase related to this technology, or as a tool for designing or creating variants (modified versions) of the glucosyltransferase related to this technology.

[0062] 3. Expression cassette and recombinant vector: The expression cassette and recombinant vector related to this technology include the DNA related to this technology as described above.

[0063] The expression cassette and recombinant vector related to this technology may contain, as necessary, transcription elements such as enhancers, CCAAT boxes, TATA boxes, and SPI sites, in addition to promoters and terminators, as regulatory factors. These regulatory factors only need to be operablely ligated to the DNA of this technology. Operable ligation means that the various regulatory factors that regulate the DNA of this technology and the DNA of this technology are ligated in a manner that allows them to function in the host cell.

[0064] Regarding the recombinant vectors related to this technology, preferred expression vectors are those constructed for genetic recombination from phages, plasmids, or viruses that can autonomously proliferate within a host. Such expression vectors are well known, and commercially available examples include pQE vectors (Qiagen Co., Ltd.), pDR540, pRIT2T (GE Healthcare Biosciences Co., Ltd.), pET vectors (Merck K.K.), and pBE-S (Takara Bio Inc.). The expression vector can be used in any combination with a host cell. For example, when using Escherichia coli as the host cell, examples include a combination of a pET vector and the DH5α strain, a combination of a pET vector and the BL21 (DE3) strain, or a combination of a pDR540 vector and the JM109 strain.

[0065] 4. Transformants The transformants related to this technology are obtained by transforming a host with the expression cassette or recombinant vector related to this technology described above.

[0066] The host used in the production of transformants according to this technology is not particularly limited as long as it has the following characteristics (1) to (4): (1) It is possible to introduce an expression cassette or recombinant vector; (2) The expression cassette or recombinant vector is stable; (3) It is capable of autonomous proliferation; and (4) It is capable of expressing the traits of the genes in the introduced expression cassette or recombinant vector. Suitable examples of such hosts include bacteria belonging to the Escherichia genus such as Escherichia coli, the Bacillus genus such as Bacillus subtilis, and the Pseudomonas genus such as Pseudomonas putida; filamentous fungi, yeast, etc. Animal cells, insect cells, plants, etc. may also be used.

[0067] Transformants of this technology can be obtained by introducing DNA related to this technology, an expression cassette related to this technology, or a recombinant vector related to this technology into a host. The site where the DNA related to this technology is introduced is not particularly limited as long as the target gene can be expressed, and may be on a plasmid or on the genome.

[0068] Specific methods for introducing the expression cassette or recombinant vector related to this technology include, for example, recombinant vector methods and genome editing methods. The conditions for introducing the expression cassette or recombinant vector into the host can be appropriately set according to the type of host, etc. If the host is bacteria, for example, methods using competent cells treated with calcium ions and electroporation can be used. If the host is yeast, for example, electroporation, spheroplast methods and lithium acetate methods can be used. If the host is animal cells, for example, electroporation, calcium phosphate methods and lipofection methods can be used. If the host is insect cells, for example, calcium phosphate methods, lipofection methods and electroporation methods can be used. If the host is plant cells, for example, electroporation, Agrobacterium methods, particle gun methods and PEG methods can be used.

[0069] Confirmation of whether the DNA related to this technology, the expression cassette related to this technology, or the recombinant vector related to this technology has been incorporated into a host can be performed by PCR, Southern hybridization, Northern hybridization, etc.

[0070] To confirm whether the DNA, expression cassette, or recombinant vector related to this technology has been incorporated into a host using PCR, for example, the genomic DNA, expression cassette, or recombinant vector can be isolated and purified from the transformant. The isolation and purification of the genomic DNA, expression cassette, or recombinant vector can be performed, for example, based on the lysate obtained by lysing the bacteria when the host is a bacterium. As a method of lysis, for example, treatment with a lytic enzyme such as lysozyme is performed, and if necessary, proteases, other enzymes, and surfactants such as sodium lauryl sulfate (SDS) are used in combination.

[0071] Furthermore, physical disruption methods such as freeze-thaw cycles and French press treatments may be combined. DNA isolation and purification from the lysate can be performed, for example, by deproteinization treatments using phenol and protease, ribonuclease treatment, alcohol precipitation, and by appropriately combining commercially available kits.

[0072] DNA cleavage can be performed according to standard methods, for example, using restriction enzyme therapy. For example, type II restriction enzymes that act on specific nucleotide sequences can be used. Binding of DNA to an expression cassette or expression vector can be performed, for example, using a DNA ligase.

[0073] Subsequently, using the separated and purified DNA as a template, primers specific to the DNA related to this technology are designed and PCR is performed. The amplified products obtained by PCR are subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide and SYBR Green solution, and the amplified products are detected as bands to confirm that transformation has occurred.

[0074] Furthermore, PCR can be performed using primers pre-labeled with fluorescent dyes or the like to detect the amplified product. In addition, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and confirmed by fluorescence and enzymatic reactions.

[0075] 5. Method for Producing Glucosyltransferase The method for producing glucosyltransferase according to this technology involves at least a culture step and a recovery step. In addition, various steps commonly used in enzyme production can be freely combined as needed. Each step will be described in detail below.

[0076] (1) Culture process The culture process is a process of culturing a microorganism that produces glucosyltransferase or a transformant related to this technology. Examples of microorganisms that produce glucosyltransferase include microorganisms of the genus Streptococcus, and an example of a microorganism of the genus Streptococcus is Streptococcus thermophilus. The microorganism used may be a wild-type strain or a mutant strain (for example, a mutant strain produced by ultraviolet irradiation).

[0077] The culture conditions in the culture process can be appropriately set considering the properties of the microorganism being cultured and the nutritional and physiological properties of the transformant being cultured. The microorganism or transformant can be cultured in solid or liquid media, but liquid culture is preferred. Furthermore, for industrial production, aerated and stirred culture is preferred.

[0078] As nutrients for the culture medium, any substance necessary for the growth of microorganisms or transformants can be used as appropriate. As a carbon source, any assimilated carbon compound is acceptable, such as glucose, sucrose, lactose, maltose, molasses, and pyruvic acid. As a nitrogen source, any assimilated nitrogen compound is acceptable, such as peptone, meat extract, yeast extract, casein hydrolysate, and soybean meal alkali extract. In addition to 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 be used as needed.

[0079] The culture temperature should be set appropriately within a range in which the microorganism or transformant can grow and produce glucosyltransferase. For example, the culture temperature can be set to 15 to 37°C.

[0080] The culture time can be, for example, 12 to 48 hours. The culture process can be completed when the glucosyltransferase reaches its maximum yield.

[0081] (2) Recovery process The recovery process is a process of recovering the glucosyltransferase related to this technology from the culture obtained in the culture process. The recovery method in the recovery process is not particularly limited, and one or more general recovery methods used in enzyme production can be freely used in combination.

[0082] Specifically, for example, if glucosyltransferase expressed using a transformant is present inside the transformant, the transformant is separated from the culture supernatant using a common separation method such as centrifugation. The culture supernatant is removed and the separated transformant is recovered. The separated transformant is treated using mechanical methods such as sonication or French press, or enzymatic methods such as lysozyme, and solubilized using enzymes such as proteases or surfactants such as sodium lauryl sulfate (SDS) as needed to obtain a water-soluble fraction containing glucosyltransferase. Alternatively, by selecting an appropriate expression cassette or expression vector and host, the expressed glucosyltransferase can be secreted into the culture medium.

[0083] The water-soluble fraction containing the glucosyltransferase obtained in this way may be subjected to purification as is, or it may be subjected to purification after concentrating a predetermined amount of glucosyltransferase in the water-soluble fraction. Concentration can be carried out by methods such as vacuum concentration, membrane concentration, salting out, or fractional precipitation using hydrophilic organic solvents (e.g., methanol, ethanol, and acetone). Furthermore, the glucosyltransferase can be purified by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0084] The purified glucosyltransferase can be powdered and distributed to the market as needed by methods such as freeze-drying, vacuum drying, or spray-drying.

[0085] 6. Method for producing one or more carbohydrates selected from α-1,3-glucan and leucose The method for producing one or more carbohydrates selected from α-1,3-glucan and leucose according to this technology includes a step of reacting sucrose with the glucosyltransferase according to this technology (hereinafter also referred to as the "enzyme action step"). In addition, a raw material preparation step, an enzyme inactivation step, a recovery step, etc. may be performed as needed. Each step will be described in detail below.

[0086] (1) Raw material preparation process The raw material preparation process is the process of preparing sucrose, which will be used as a raw material. The sucrose used as a raw material may be in the form of a composition mixed with other components, as long as it does not impair the function or effect of this technology.

[0087] (2) Enzyme action step The enzyme action step is a step in which the glucosyltransferase according to this technology is applied to the sucrose, which is the raw material. The glucosyltransferase according to this technology may be used alone or in combination of two or more types.

[0088] Furthermore, as long as the action and effects of this technology are not impaired, it may be used in combination with other enzymes other than the glucosyltransferase related to this technology, depending on the purpose.

[0089] When using two or more enzymes in combination, the two or more enzymes may be added to the sucrose raw material simultaneously or separately. The order in which the two or more enzymes are added separately is not particularly limited. Details of the glucosyltransferase related to this technology are as described above and will therefore not be explained here.

[0090] The various conditions of the enzyme action process can be freely set, as long as they do not impair the effectiveness of this technology. For example, pH, temperature, action time, etc., can be set according to the physicochemical properties of the enzyme used, such as the optimal pH, stable pH range, optimal temperature, and temperature stability. The optimal reaction conditions can be determined through preliminary experiments. The following are examples of conditions for using each enzyme.

[0091] When the glucosyltransferase according to this technology is applied to the sucrose used as a raw material, the pH can be set to, for example, pH 2.0 to 9.0, preferably pH 2.5 to 8.0, and more preferably pH 3.0 to 6.0.

[0092] The temperature at which the glucosyltransferase according to this technology is applied to the sucrose raw material can be set to, for example, 10°C to 70°C, preferably 20°C to 50°C, and more preferably 25°C to 45°C.

[0093] When the glucosyltransferase according to this technology is applied to the sucrose used as a raw material, the application time can be set to, for example, 15 minutes to 120 hours, preferably 30 minutes to 72 hours, and more preferably 2 hours to 48 hours.

[0094] The amount of glucosyltransferase relative to sucrose in the enzymatic action step can be freely set as long as it does not impair the effects of this technology. The lower limit of the amount of glucosyltransferase per 1 g of sucrose can be set to, for example, 0.1 U or more, and from the viewpoint of further enhancing the α-1,3-glucan production effect and the sucrose reduction effect, it can be preferably set to 0.5 U or more, more preferably 1 U or more, 5 U or more, and even more preferably 10 U or more.

[0095] The upper limit for the amount of glucosyltransferase per 1 g of sucrose can be set to, for example, 450 U or less, 90 U or less, 45 U or less, or 30 U or less.

[0096] In this technology, glucosyltransferase activity is defined as the amount of enzyme that hydrolyzes 1 μmol of sucrose per minute when treated with a 50 mM sucrose solution (50 mM potassium phosphate buffer, pH 7.0) at 30°C, with 1 unit (1 U) being the amount of enzyme.

[0097] (3) Enzyme Inactivation Step The enzyme inactivation step is a step in which the enzyme is inactivated after the enzyme action step described above. The enzyme inactivation step is not an essential step and can be performed as needed. Furthermore, if multiple enzymes are used in the enzyme action step described above, enzyme inactivation can be performed between the actions of the enzymes. For example, if multiple enzymes are used in the enzyme action step, it is possible to perform the enzyme inactivation step as appropriate in the middle of the enzyme action step.

[0098] The method for inactivating an enzyme is not particularly limited, and one or more general inactivation methods can be freely combined and used depending on the properties of the enzyme to be inactivated. For example, one method is to inactivate the enzyme by heating it above its enzyme inactivation temperature.

[0099] (4) Recovery Process The recovery process is a process for recovering the manufactured α-1,3-glucan. Depending on the type of α-1,3-glucan to be manufactured, one or more general recovery methods used in the manufacture of α-1,3-glucan can be freely combined and used.

[0100] The recovered α-1,3-glucan can be further processed, etc., depending on the form of the final product, as long as it does not impair the function or effect of this technology.

[0101] 7. Method for Manufacturing Food and Beverages, and Method for Reducing Sugars in Food and Beverages The method for manufacturing food and beverages according to this technology, and the method for reducing sugars in food and beverages according to this technology, include a step of acting on sugar-containing food and beverages with the glucosyltransferase according to this technology (hereinafter also referred to as the "enzyme action step"). In addition, a raw material preparation step, an enzyme inactivation step, a recovery step, etc., may be performed as needed. Each step will be described in detail below.

[0102] (1) Raw material preparation process The raw material preparation process is the process of preparing sugar-containing food and beverages or their raw materials to be used as raw materials. The raw materials that can be used in this technology are not particularly limited in terms of origin, type, etc., as long as they do not impair the effects of this technology, and can be freely selected according to the target food and beverage. In this technology, raw materials containing sucrose (sucrose-containing raw materials) are particularly preferred.

[0103] The origin of the raw materials is not particularly limited as long as it does not involve the action or effect of this technology, and can be freely selected. Examples of raw materials for fruit juice used in fruit juice beverages include orange (including mandarin), pineapple, grapefruit, apple, grape, peach, strawberry, banana, mango, melon, apricot, lemon, kiwi, blueberry, pear, pomegranate, lime juice, and other fruit juices listed in the Fruit Beverage Quality Labeling Standards.

[0104] Furthermore, examples of vegetable juices used as raw materials for the vegetable juice beverages of this technology include those from carrots, spinach, onions, tomatoes, celery, bell peppers, pumpkins, corn, carrots, cucumbers, beets, parsley, lettuce, cauliflower, broccoli, kale, red bell peppers, ginger, green peppers, and red cabbage.

[0105] Examples of raw material forms include liquid, paste, and slurry, with liquid being more preferable. Furthermore, the sugar-containing food and beverages used in this technology, or the raw materials that can be used in food and beverages related to this technology, can be used as is, or in liquid, paste, or slurry form. Preferably, they can also be used in the form of fresh juice, concentrated juice, paste-like juice, powdered juice, etc.

[0106] (2) Enzyme action step The enzyme action step is a step in which sugar-containing food and beverages and / or their raw materials are treated with the glucosyltransferase related to this technology. The details of the enzyme action step are the same as those of the enzyme action step in the method for producing α-1,3-glucan described above, so the explanation is omitted here.

[0107] (3) Enzyme Inactivation Step The enzyme inactivation step is a step in which the enzyme is inactivated after the enzyme action step described above. The details of the enzyme inactivation step are the same as the enzyme inactivation step in the method for producing α-1,3-glucan described above, so the explanation is omitted here.

[0108] (4) Recovery Process The recovery process is the process of recovering the manufactured food and beverages. Depending on the type of food and beverage being manufactured, one or more general recovery methods used in the manufacture of food and beverages can be freely combined and used.

[0109] The collected food and beverages may be further processed, etc., depending on the form of the final product, to the extent that the function and effect of this technology are not impaired.

[0110] 8. Food and beverages: Food and beverages relating to this technology are characterized by being obtained using the glucosyltransferase relating to this technology. Details of the glucosyltransferase relating to this technology are as described above and will therefore not be explained here.

[0111] (1) Amount of sugars contained in food and beverages The amount of sugars contained in food and beverages used in this technology is not particularly limited, but it is preferable that it is reduced compared to the amount of sugars contained in food and beverages manufactured without using the glucosyltransferase according to this technology. Specifically, the total amount of sugars contained in food and beverages according to this technology is, for example, 98% or less, preferably 97% or less, more preferably 96% or less, even more preferably 95% or less, 90% or less, or 85% or less compared to the total amount of sugars contained in food and beverages manufactured without using the glucosyltransferase according to this technology.

[0112] The sugar reduction rate of food and beverages produced using this technology compared to food and beverages produced without using the glucosyltransferase according to this technology is, for example, 2% or more, preferably 3% or more, more preferably 4% or more, even more preferably 5% or more, 10% or more, or 15% or more.

[0113] In particular, it is preferable that the amount of sucrose contained in the food and beverages according to this technology is reduced compared to the sucrose content in food and beverages manufactured without using the glucosyltransferase according to this technology. Specifically, the sucrose content in the food and beverages according to this technology is, for example, 50% or less, preferably 40% or less, more preferably 30% or less, even more preferably 20% or less, and even more preferably 10% or less, compared to the sucrose content in food and beverages manufactured without using the glucosyltransferase according to this technology.

[0114] The sucrose reduction rate of food and beverages according to this technology compared to food and beverages produced without using the glucosyltransferase according to this technology is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more, or 95% or more.

[0115] (2) Types of food and beverages The types of food and beverages related to this technology are not particularly limited as long as they do not impair the effects of this technology, but beverages are preferred. Examples of beverages include vegetable juices and fruit juices. Other examples of foods include Japanese sweets (mochi, manju, daifuku, castella, anko, etc.), Western sweets (cream, cake, ice cream, etc.), soups, sauces, dressings, etc.

[0116] 9. Enzyme preparation for sugar reduction The enzyme preparation for sugar reduction related to this technology is an enzyme preparation containing the glucosyltransferase related to this technology. Details of the glucosyltransferase related to this technology are as described above and will therefore not be explained here.

[0117] (1) Glucosyltransferase content The glucosyltransferase content in the enzyme preparation for sugar reduction according to this technology can be freely set as long as it does not impair the effects of this technology. The lower limit of the glucosyltransferase content can be set to, for example, 0.1 U or more per 1 g of sucrose in the food and beverage to be reduced in sugars, and from the viewpoint of further enhancing the sugar reduction effect, it can be preferably set to 0.5 U or more, more preferably 1 U or more, 5 U or more, and even more preferably 10 U or more.

[0118] The upper limit of the glucosyltransferase content is not particularly limited as long as it does not impair the effectiveness of this technology, but it can be set to, for example, 450 U or less, 90 U or less, 45 U or less, or 30 U or less per gram of sucrose in the food and beverages targeted for sugar reduction.

[0119] (2) Other Components The enzyme preparation for sugar reduction related to this technology may be used in combination with other components, as long as the action and effect of this technology are not impaired. Other components may include, for example, excipients, pH adjusters, colorants, flavoring agents, disintegrants, lubricants, stabilizers, enzymes, and other components commonly used in pharmaceutical formulation. Furthermore, known or future functional components may be used in combination as appropriate for the purpose.

[0120] The present technology will be described in more detail below based on the following examples. The examples described below are representative examples of the present technology and should not be interpreted as narrowing the scope of the present technology.

[0121] 1. Selection of Microorganisms: In order to find novel glucosyltransferases, a screening was conducted using glucosyltransferase activity as an indicator from a library of bacterial strains. As a result, microorganisms of the genus Streptococcus were selected as promising candidates.

[0122] 2. Sequence Verification: The amino acid sequences (SEQ ID NOs: 1 and 2) of glucosyltransferase derived from Streptococcus thermophilus were determined from genomic DNA information obtained through genomic DNA analysis using a next-generation sequencer.

[0123] 3. Construction of the Expression System: The target sequence was incorporated using a commercially available expression vector. PCR and infusion reactions were performed using primers. An expression plasmid containing the target sequence was constructed using E. coli strain BL21 (DE3), and transformants (heteroexpressing glucosyltransferase derived from Streptococcus thermophilus) were obtained using the heat shock method. Sequence analysis of the obtained transformants was performed to confirm whether the target sequence was incorporated.

[0124] 4. Culture of Transformants The transformants obtained above (Streptococcus thermophilus glucosyltransferase heterologously expressing strain) were pre-cultured by inoculating one colony from agar medium into 4 mL of ampicillin-containing LB liquid medium (50 μg / mL) in a test tube and culturing at 37°C, ON, and 160 rpm. For the main culture, 1 mL of the pre-culture solution was inoculated into 100 mL of ampicillin-containing EAR fresh medium (50 μg / mL) in a 500 mL Sakaguchi flask and culturing at 37°C, 24 hours, and 160 rpm. The cultures were then collected.

[0125] 5. Purification of Glucosyltransferase A crude solution of glucosyltransferase was obtained from the culture obtained above by affinity chromatography. This crude solution was subjected to a column equilibrated with 100 mM potassium phosphate buffer (pH 7.5) + 0.5 M sodium chloride and 40 mM imidazole ("Hitrap"). TM The column was subjected to a 5 mL Butyl FF (Cytiva) buffer. After washing the column with the same buffer, the glucosyltransferase adsorbed to the column was eluted with 100 mM potassium phosphate buffer (pH 7.5) + 0.5 M sodium chloride, 300 mM imidazole (0% to 50%, 65 mL), and then fractionated. Glucosyltransferase activity was confirmed in a portion of each obtained fraction using the method described later, and the fraction exhibiting glucosyltransferase activity was collected to obtain a purified glucosyltransferase solution. The purified fraction was desalted and concentrated 10-fold using Amicon 10K. Purification was confirmed by SDS-PAGE. The molecular weight was 168 kDa.

[0126] 6. As a substrate for measuring glucosyltransferase activity, 1700 μL of 50 mM potassium phosphate buffer (pH 7.0), 100 μL of 1 M sucrose solution (50 mM (1.71%) (17.1 g / L)), and 200 μL of enzyme solution were added to a test tube and mixed, and the mixture was reacted at 30°C for 1 hour. After the reaction, the amount of free fructose was measured using E-Kit Liquid D-glucose / fructose (manufactured by J.K. International Co., Ltd.), and the activity was calculated as a relative value. One unit (1 U) of enzyme was defined as the amount that hydrolyzes 1 μmol of sucrose per minute when treated with 50 mM sucrose solution (50 mM potassium phosphate buffer (pH 7.0)) as the substrate at 30°C.

[0127] 7. Characterization of Glucosyltransferase Activity was measured in the same manner as described above, except that the enzyme reaction temperature was changed to 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C, and the activity was calculated as a relative value. The results are shown in Figure 1. As shown in Figure 1, the temperature at which the glucosyltransferase obtained from the heterologous expression strain of glucosyltransferase derived from Streptococcus thermophilus showed the highest activity in the 30-60°C range was 30°C.

[0128] 8. Examination of the applications of glucosyltransferase (1) Materials used The materials used are shown in Table 1 below.

[0129]

[0130] (2) Experimental method 800 μL (925.5 μL if enzyme 2 is added) of the materials in Table 1 into a 1.5 mL tube, and enzyme 1 (glucosyltransferase containing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1) or enzyme 2 (glucosyltransferase containing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2) was added in the amounts shown in Table 3 below. After stirring by inversion, the pH was adjusted to the levels shown in Table 3 below as needed, and the mixture was reacted at 30°C for 24 hours. After the reaction, the amount of each sugar was analyzed using HPLC under the conditions shown in Table 2 below. A control was used for the case where no enzyme was added.

[0131]

[0132] (3) Results The results are shown in Table 3 below.

[0133]

[0134] (4) Discussion As shown in Table 3, it was found that when enzyme 1 or 2 was applied to food and beverages, the amount of sugars in the food and beverages was reduced. Among the sugars, the reduction effect on sucrose was particularly significant. Furthermore, when enzyme 1 or 2 was applied to food and beverages, the production of heptasaccharides or more, including leucose; soluble α-1,3-glucan; nigerooligosaccharides such as nigeroose, nigerotriose, nigerotetraose, nigeropentaose, and nigerohexaose; or insoluble α-1,3-glucan, was confirmed.

Claims

1. Glucosyltransferases comprising a polypeptide as shown in any of (1) to (4) below: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (2) a polypeptide having glucosyltransferase activity, wherein one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in SEQ ID NO: 1 or 2; (3) a polypeptide having glucosyltransferase activity, wherein the amino acid sequence shown in SEQ ID NO: 1 has a sequence identity of 93% or more to the amino acid sequence shown in SEQ ID NO: 1; and (4) a polypeptide having glucosyltransferase activity, wherein the amino acid sequence shown in SEQ ID NO: 2 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO:

2.

2. The glucosyltransferase according to claim 1, which is derived from a microorganism of the genus Streptococcus.

3. The glucosyltransferase according to claim 2, which is derived from Streptococcus thermophilus.

4. DNA comprising any of the following polynucleotides (1) to (3): (1) a polynucleotide encoding the glucosyltransferase described in claim 1; (2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 or 6; (3) a polynucleotide encoding a polypeptide having glucosyltransferase activity, comprising a sequence equivalent to the base sequence shown in SEQ ID NO: 5 or 6.

5. An expression cassette or recombinant vector comprising the DNA described in claim 4.

6. A transformant obtained by transforming a host with the expression cassette or recombinant vector described in claim 5.

7. A method for producing glucosyltransferase, comprising: a culture step of culturing a microorganism of the genus Streptococcus or a transformant according to claim 6; and a recovery step of recovering the glucosyltransferase according to claim 1 from the culture obtained in the culture step.

8. A method for producing one or more carbohydrates selected from α-1,3-glucan and leucrose, comprising the step of reacting sucrose with the glucosyltransferase described in claim 1.

9. A method for producing food and beverages, comprising the step of reacting a sugar-containing food and beverage and / or its raw materials with the glucosyltransferase described in claim 1.

10. A method for reducing sugars in food and beverages, comprising the step of reacting sugar-containing food and beverages and / or their raw materials with the glucosyltransferase described in claim 1.

11. Food and beverages obtained using the glucosyltransferase described in claim 1.

12. An enzyme preparation for reducing sugars, comprising the glucosyltransferase described in claim 1.

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