Method for producing rebaudioside d and rebaudioside m

Abstract

To provide a method for producing rebaudioside D and rebaudioside M by reaction of glycosyltransferase, and to provide a composition for producing rebaudioside D and rebaudioside M containing glycosyltransferase.SOLUTION: Provided is a method for preparing rebaudioside D, the method including preparing rebaudioside D by reacting glucose-linked nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B), wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins consisting of an amino acid sequence represented by a specific sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present application relates to a method for producing rebaudioside D and rebaudioside M by a glycosyltransferase reaction, and a composition for producing rebaudioside D and rebaudioside M, which contains a glycosyltransferase. [Background technology]

[0002] The World Health Organization (WHO) has recommended reducing daily sugar intake due to the risk of diseases (obesity) caused by sugar intake, and various government-led policies to reduce sugar intake are being actively discussed, particularly in developed countries. Furthermore, there is an increasing need in the market for various alternative sweeteners to replace sugar and high fructose, and the development and commercialization of alternative sweeteners continues.

[0003] Alternative sweeteners include synthetic sweeteners (such as saccharin, aspartame, and sucralose), synthetic sugar alcohols, and The sweeteners have been evolving from sugars (maltitol, xylitol) to high-intensity sweeteners (rebaudioside A, liquorice). However, despite growing consumer demand for natural sweeteners due to concerns about the long-lasting stability of synthetic sweeteners, natural sweeteners have limitations in terms of taste quality, including the off-flavor and odor inherent to natural sweeteners, and are therefore unable to fully replace traditional low-calorie and zero-calorie products that primarily use synthetic sweeteners.

[0004] In this regard, a natural high-power sweetener that has attracted much attention in recent years is stevia sweetener extracted from stevia leaves. Stevia sweetener has been reported to be calorie-free, have beneficial effects on blood glucose and insulin levels, and have no side effects on the human body, and has potential as an alternative sweetener. However, it has the drawback of being very bitter, which limits its use in reducing sugar intake.

[0005] Stevia is a perennial plant of the Asteraceae family native to Paraguay in South America, and its scientific name is Stevia rebaudiana Bertoni. Stevia contains compounds that are 200 to 300 times sweeter than sugar, and these sweet compounds are extracted and used as natural sweeteners. The sweet compounds in stevia extract include various steviol glycosides, such as stevioside, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside E.

[0006] Among the sweetening components of stevia extract, stevioside (STV), rebaudioside A (Reb A), and rebaudioside C (Reb C) are found in relatively high amounts in stevia leaves and are extracted and refined to a high degree for industrialization and commercialization. However, they have the drawback of being very bitter, which limits their use in reducing sugar intake.

[0007] On the other hand, rebaudioside D (Reb D) and rebaudioside M (Reb M) have less bitterness and superior sweetness quality than STV, rebaudioside A, and rebaudioside C, making them valuable as alternative sweeteners. However, rebaudioside D and rebaudioside M are present in extremely small amounts in stevia leaves, and the method of extracting and purifying them from the leaves is costly. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Registration No. 1404728 [Patent Document 2] International Publication No. 2014 / 133248 [Non-patent literature]

[0009] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0010] The present applicant has conducted extensive research to develop an enzyme having the activity of converting rebaudioside A to rebaudioside D, and as a result has found that a polypeptide sequence whose glycosyltransferase activity has not previously been disclosed has glycosyltransferase activity. The applicant has confirmed that the polypeptide has the glycosyltransferase activity of converting rebaudioside A to rebaudioside D, and has completed the present application. [Means for solving the problem]

[0011] An object of the present application is to provide a method for producing rebaudioside D, comprising the step of reacting a glucose-bound nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B), which is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, to produce rebaudioside D.

[0012] Another object of the present application is to provide a method for producing rebaudioside M, comprising the steps of reacting a glucose-bound nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B) to produce rebaudioside D, and reacting the rebaudioside D with a glucose-bound nucleotide diphosphate in the presence of glycosyltransferase A (UGT-A) to produce rebaudioside M, wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3.

[0013] Another object of the present application is to provide a method for producing rebaudioside D from rebaudioside A, comprising the step of producing rebaudioside D by reacting sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase, and glycosyltransferase B (UGT-B) in a single reaction system.

[0014] Further, the present application provides a method for the production of sucrose, nucleotide diphosphates, rebaudioside A, rebaudioside D, sucrose synthase, glycosyltransferase A (UGT-A) and glycosyltransferase B ( The present invention provides a method for producing rebaudioside M from rebaudioside A, comprising the step of reacting rebaudioside A with rebaudioside B (UGT-B) in the same reaction system to produce rebaudioside M.

[0015] Another object of the present application is to provide a composition for producing rebaudioside D, which contains the glycosyltransferase B (UGT-B).

[0016] Another object of the present application is to provide a composition for producing rebaudioside M, which comprises glycosyltransferase A (UGT-A) and glycosyltransferase B (UGT-B).

[0017] Furthermore, the present application aims to provide use of glycosyltransferase B (UGT-B), which is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, as a glycosyltransferase that converts rebaudioside A to rebaudioside D. [Effects of the Invention]

[0018] The method for producing rebaudioside D and rebaudioside M using glycosyltransferase B (UGT-B) according to the present invention can provide rebaudioside D and rebaudioside M in high purity and high yield with almost no by-products, is economical because it uses inexpensive raw materials, has a simple procedure with little time consumption, and is useful for mass production of rebaudioside D and rebaudioside M. [Brief explanation of the drawings]

[0019] [Figure 1] 1 shows the results of HPLC analysis demonstrating that rebaudioside A is converted to rebaudioside D by glycosyltransferase B (UGT-B_6). [Figure 2] 1 shows the results of HPLC analysis demonstrating that rebaudioside A is converted to rebaudioside D, rebaudioside D isomers, and rebaudioside M isomers by glycosyltransferase B (UGT-B_7). [Figure 3] 1 shows the results of HPLC analysis demonstrating the conversion of rebaudioside A to rebaudioside D by glycosyltransferase B (UGT-B_8). [Figure 4] 1 shows the results of HPLC analysis demonstrating the production of rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside A from stevioside by glycosyltransferase B (UGT-B_6), glycosyltransferase A (UGT-A), and sucrose synthase. [Figure 5]1 shows the results of HPLC analysis demonstrating the production of rebaudioside M isomers, rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside A from stevioside by glycosyltransferase B (UGT-B_7), glycosyltransferase A (UGT-A), and sucrose synthase. [Figure 6] 1 shows the results of HPLC analysis demonstrating the production of rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside A from stevioside by glycosyltransferase B (UGT-B_8), glycosyltransferase A (UGT-A), and sucrose synthase. [Figure 7] 1 is an HPLC analysis showing the conversion of rebaudioside D to rebaudioside M by glycosyltransferase A (UGT-A). [Figure 8] 1 shows the results of LC-MS / MS analysis of rebaudioside D isomers. [Figure 9] 1 shows the results of LC-MS / MS analysis of rebaudioside D. [Figure 10] 1 shows the results of LC-MS / MS analysis of rebaudioside M isomers. [Figure 11] 1 shows the results of LC-MS / MS analysis of rebaudioside M. DETAILED DESCRIPTION OF THE INVENTION

[0020] These will be specifically described below. Note that each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. In other words, various elements disclosed in this application Any combination of the above is included in this application. Furthermore, this application is not limited to the specific descriptions below. Furthermore, many papers and patent documents are referenced throughout this specification, and citations are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0021] One aspect of the present application provides a method for producing rebaudioside D, comprising the step of reacting a glucose-bound nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B), which is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, to produce rebaudioside D.

[0022] In this application, "glycosyltransferase (UDP (Uridine diphosphate)-glycosyltransferase, UGT)" "Glycosyltransferase" refers to an enzyme that has the activity of transferring a monosaccharide moiety from a glycosyl donor to a glycosyl acceptor molecule, specifically an enzyme that uses a UDP-sugar as a glycosyl donor. In this application, the term "glycosyltransferase" is also used interchangeably with "UDP-glycosyltransferase" and "UGT."

[0023] The glycosyltransferase may be produced from recombinant Escherichia coli, Bacillus, yeast, Corynebacterium, or Agrobacterium transformed with a vector containing a glycosyltransferase gene, or may be produced from the above-mentioned Escherichia coli or the like and further purified, or may be purchased as a commercially available product, but is not limited to these. Furthermore, the glycosyltransferase is known in the art, and the protein and gene sequences of the glycosyltransferase can be obtained from known databases, such as, but not limited to, GenBank of NCBI.

[0024] In the present application, it has been discovered that glycosyltransferase B (UGT-B), which is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, has the enzymatic activity of converting rebaudioside A to rebaudioside D, and a new use of UGT-B is provided.

[0025] Specifically, the glycosyltransferase B (UGT-B) of the present application may have the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO: 3, or may contain the amino acid sequence, or may essentially consist of the amino acid sequence, It may consist of an amino acid sequence.

[0026] Furthermore, the glycosyltransferase B (UGT-B) of the present application may comprise an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, or the amino acid sequence represented by SEQ ID NO: 3. Furthermore, it goes without saying that the present application also includes glycosyltransferase B (UGT-B) having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as the amino acid sequence has such homology or identity and exhibits an effect equivalent to that of the glycosyltransferase B (UGT-B) of the present application.

[0027] Even if the present application describes a "polypeptide or protein comprising an amino acid sequence represented by a specific SEQ ID NO," "a polypeptide or protein consisting of an amino acid sequence represented by a specific SEQ ID NO," or "a polypeptide or protein having an amino acid sequence represented by a specific SEQ ID NO," a partial sequence may be deleted, modified, substituted, conservatively substituted, or added, as long as it has the same or equivalent activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO. It goes without saying that proteins having a modified amino acid sequence are also used in the present application. For example, addition of a sequence at the N-terminus and / or C-terminus of the amino acid sequence that does not change the function of the protein, naturally occurring mutations, silent mutations, etc. or those having conservative substitutions.

[0028] For example, the amino acid sequence may have an addition or deletion of a sequence at the N-terminus, C-terminus and / or internally, a naturally occurring mutation, a silent mutation, or a conservative substitution that does not alter the function of the glycosyltransferase B (UGT-B) of the present application.

[0029] In this application, a "conservative substitution" means that an amino acid is "Amino acid substitution" refers to substitution with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids are further classified into those with electrically charged side chains and those with uncharged side chains. Amino acids with uncharged side chains are further classified as nonpolar amino acids and polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, and leucine. Conservative substitutions include cysteine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Conservative substitutions typically have little or no effect on the activity of the resulting polypeptide. Conservative substitutions typically have little or no effect on the activity of a protein or polypeptide.

[0030] The glycosyltransferase B (UGT-B) may also contain amino acid deletions or additions that minimally affect the properties and secondary structure of the polypeptide. For example, the polypeptide may be linked to an N-terminal signal (or leader) sequence of a protein involved in co- or post-translational protein transfer. The polypeptide may also be linked to other sequences or linkers that allow for identification, purification, or synthesis of the polypeptide.

[0031] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0032] Conserved polynucleotide or polypeptide sequence homology or identity Identity may be determined by standard alignment algorithms, with default gap penalties established by the program being used. Substantially homologous or identical sequences are detected under moderate or high stringency conditions. Generally, under stringent conditions, the hybridization will hybridize to the entire sequence or a portion thereof. It goes without saying that hybridization also includes hybridization to polynucleotides having common codons or codons that take into account codon degeneracy in polynucleotides.

[0033] Any two polynucleotide or polypeptide sequences that have homology, similarity, or identity Whether or not a sequence matches can be determined using a known computer algorithm, such as the "FASTA" program, with default parameters as described in, for example, Non-Patent Document 1. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, it can be determined using BLAST or Clustal W from the National Center for Biotechnology Information. Homology, similarity or identity can be determined.

[0034] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in Non-Patent Document 8. Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program are (1) a binary comparison matrix (identity takes a value of 1, non-identity a value of 0) and (2) a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) as disclosed in Non-Patent Document 9). (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0035] As an example of the present application, the glycosyltransferase B (UGT-B) of the present application may have the activity of converting rebaudioside A to rebaudioside D.

[0036] "Corresponding to," in this application, means the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid of the sequence to which the particular sequence refers. In this application, a "corresponding region" generally refers to a region in a related or reference protein. "A" means a similar or corresponding position.

[0037] For example, if any amino acid sequence is aligned with SEQ ID NO: 1, Based on this, each amino acid residue in the amino acid sequence can be numbered with reference to the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the amino acid positions or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared with a query sequence (also referred to as a "reference sequence").

[0038] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 3) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) can be used, but the method is not limited to these. Any sequence alignment program or pairwise sequence comparison algorithm known in the art can be used as appropriate. It is possible.

[0039] The polynucleotide encoding glycosyltransferase B (UGT-B) of the present application is not only a base sequence encoding the amino acids represented by each of the above SEQ ID NOs, but also a base sequence showing 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more homology with the above sequence, and is substantially identical or similar to each of the above proteins. Any gene sequence that encodes a protein that exhibits the corresponding efficacy may be used. Needless to say, the present application also includes base sequences with such homology, including base sequences in which a portion of the sequence has been deleted, modified, substituted, or added.

[0040] In this application, a "polynucleotide" refers to a polymer consisting of covalently bonded nucleotide monomers. A polymer of nucleotides linked together in a long chain by cleavage, It refers to a longer DNA or RNA chain, and more specifically to a polynucleotide fragment encoding the glycosyltransferase B (UGT-B).

[0041] The polynucleotide encoding glycosyltransferase B (UGT-B) of the present application may comprise a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. As an example of the present application, the polynucleotide may have or comprise the sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Furthermore, the polynucleotide may consist of the sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or may essentially consist of the sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0042] The polynucleotide of the present application can be modified in various ways in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the glycosyltransferase B (UGT-B) of the present application is to be expressed, as long as the amino acid sequence of the glycosyltransferase B (UGT-B) of the present application is not changed. Specifically, the polynucleotide of the present application has a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or contains said base sequence; or consists of a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or is essentially composed of said base sequence, but is not limited to these.

[0043] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. "Conditions" refer to conditions under which hybridization is possible. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, these conditions include conditions under which polynucleotides with high homology or identity, e.g., polynucleotides having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional southern hybridization, i.e., 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0044] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0045] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0046] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0047] In one example of the present application, the glucose-linked nucleotide diphosphate is prepared by reacting sucrose with nucleotide diphosphate in the presence of sucrose synthase, but is not limited thereto.

[0048] The term "sucrose synthase" in the present application refers to an enzyme that plays a role in the metabolism of a plant body in reversibly transferring glucose bound to a nucleotide diphosphate to fructose to produce sucrose, and in the present application, it exhibits the activity of reacting sucrose with a nucleotide diphosphate in the pH range of 5 to 10 and separating them into a nucleotide diphosphate bound to a glucose and fructose.

[0049] The sucrose synthase is derived from rice, corn, wheat, bamboo, Arabidopsis thaliana, turfgrass, barley, sorghum, or potato, and is preferably derived from rice, corn, wheat, or barley, and more preferably from rice, particularly Oryza sativa. The sucrose synthase may be one produced from recombinant Escherichia coli, Bacillus, yeast, Corynebacterium, or Agrobacterium transformed with a vector having a sucrose synthase gene, one produced from the above-mentioned Escherichia coli or the like and further purified, a sucrose synthase known in the art, or a commercially available sucrose synthase, but is not limited to these.

[0050] Specifically, the sucrose synthase of the present application may have the amino acid sequence shown in SEQ ID NO: 5, may include the amino acid sequence, or may essentially consist of the amino acid sequence, It may consist of an amino acid sequence.

[0051] The sucrose may be any substance that can act as a substrate for sucrose synthase to supply glucose to nucleotide diphosphate, and examples thereof include, but are not limited to, raw sugar or sugar.

[0052] In the present application, the nucleotide diphosphate may be a purine nucleotide or a pyrimidine nucleotide, and is preferably, but not limited to, uridine diphosphate.

[0053] The glucose-linked nucleotide diphosphate may be reacted with rebaudioside A to produce rebaudioside D by glycosyltransferase B (UGT-B) of the present application.

[0054] In one embodiment of the present application, rebaudioside A was prepared by reacting a glucose-linked nucleotide diphosphate with stevioside in the presence of glycosyltransferase A (UGT-A). However, it is not limited to this.

[0055] The glycosyltransferase A (UGT-A) may react a glucose-linked nucleotide diphosphate with stevioside to produce rebaudioside A.

[0056] The glycosyltransferase A (UGT-A) is derived from Oryza sativa, Stevia rebaudiana Bertoni, Bambusa oldhamii, Brachypodium distachyon, Hordeum vulgare, Sorghum b The glycosyltransferase may be derived from Bacillus icolor, Zea mays, or Arabidopsis thaliana. The glycosyltransferase is preferably derived from Oryza sativa, Stevia rebaudiana Bertoni, or Bambusa oldhamii. The glycosyltransferase is more preferably derived from Stevia rebaudiana Bertoni. The glycosyltransferase A (UGT-A) may be produced from recombinant Escherichia coli, Bacillus, yeast, Corynebacterium, or Agrobacterium transformed with a vector having a glycosyltransferase gene, or produced from the above-mentioned Escherichia coli or the like and further purified, or may be a glycosyltransferase known in the art, or commercially available, but is not limited to these.

[0057] Specifically, the glycosyltransferase A (UGT-A) of the present application may have the amino acid sequence shown in SEQ ID NO: 4, may contain the amino acid sequence, or may be essentially composed of the amino acid sequence. , or may consist of the amino acid sequence.

[0058] The stevioside is a hot water or aqueous ethanol extract of Stevia rebaudiana, or a purified product thereof, or a by-product of rebaudioside A production from the extract, and the stevioside content is 10% by weight or more, preferably 50% by weight or more, more preferably 70% by weight or more, and even more preferably 80% by weight or more of the total weight of steviol glycosides, but is not limited thereto.

[0059] In one embodiment of the present application, rebaudioside D is prepared as shown in Reaction Scheme 1, but is not limited thereto.

[0060] [Reaction Scheme 1] TIFF2026032151000002.tif17127

[0061] Specifically, the production method may be carried out continuously in the same reaction system.

[0062] In this application, "same reaction system" means that reactions occur sequentially in one reaction system or reaction system.

[0063] The production method of the present application provides a continuous reaction system for synthesizing rebaudioside A in high yield by specifically binding one glucose to the C-3' position of 13-O-glucose of stevioside according to Reaction Scheme 1, and synthesizing rebaudioside D from rebaudioside A.

[0064] In one embodiment of the present application, the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, and further produces an isomer of rebaudioside D, but is not limited thereto.

[0065] For example, rebaudioside D has the following structural formula, but is not limited thereto:

[0066] [ka]

[0067] Another aspect of the present application provides a method for producing rebaudioside M, comprising the steps of reacting a glucose-linked nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B) to produce rebaudioside D, and reacting the rebaudioside D with a glucose-linked nucleotide diphosphate in the presence of glycosyltransferase A (UGT-A) to produce rebaudioside M, wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3.

[0068] The nucleotide diphosphates, glycosyltransferase B (UGT-B), rebaudioside A, and rebaudioside D are as described above.

[0069] In one embodiment of the present application, the glucose-linked nucleotide diphosphate is prepared by reacting sucrose with nucleotide diphosphate in the presence of sucrose synthase, as shown in Reaction Scheme 1, but is not limited thereto.

[0070] Specifically, the sucrose synthase of the present application may have the amino acid sequence shown in SEQ ID NO: 5, or may include the amino acid sequence. It may be essentially consisting of the sequence, It may consist of an amino acid sequence.

[0071] In one embodiment of the present application, rebaudioside A is prepared by reacting a glucose-linked nucleotide diphosphate with stevioside in the presence of glycosyltransferase A (UGT-A) as shown in Reaction Scheme 1, but is not limited thereto.

[0072] Specifically, the glycosyltransferase A (UGT-A) may have the amino acid sequence shown in SEQ ID NO: 4, may contain the amino acid sequence, or may essentially consist of the amino acid sequence. It may consist of the following amino acid sequence.

[0073] The glycosyltransferase A (UGT-A) of the present application may react a glucose-linked nucleotide diphosphate with rebaudioside D to produce rebaudioside M.

[0074] Specifically, the glycosyltransferase A (UGT-A) of the present application may have the amino acid sequence shown in SEQ ID NO: 4, may contain the amino acid sequence, or may be essentially composed of the amino acid sequence. , or may consist of the amino acid sequence.

[0075] In one embodiment of the present application, the rebaudioside M is prepared as shown in Reaction Scheme 2, but is not limited thereto.

[0076] [Reaction Scheme 2] TIFF2026032151000004.tif14143

[0077] Specifically, the production method may be carried out continuously in the same reaction system.

[0078] The production method of the present application provides a continuous reaction system for synthesizing rebaudioside A from stevioside in high yield, synthesizing rebaudioside D from rebaudioside A, and synthesizing rebaudioside M from rebaudioside D, according to Reaction Scheme 2.

[0079] Therefore, the production method of the present application uses easily available and inexpensive raw materials such as stevioside and rebaudioside A, and can convert bitter components such as stevioside and rebaudioside A contained in stevia extract into rebaudioside D and rebaudioside M, which are palatable components, and is therefore useful for producing stevia sweeteners with good sweetness quality.

[0080] In one embodiment of the present application, the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, and further produces an isomer of rebaudioside M, but is not limited thereto.

[0081] For example, rebaudioside M has the following structural formula, but is not limited thereto:

[0082] [ka]

[0083] Yet another aspect of the present application provides a method for producing rebaudioside D from rebaudioside A, comprising the step of producing rebaudioside D by reacting sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase, and glycosyltransferase B (UGT-B) in a reaction system, wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3.

[0084] The sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase, glycosyltransferase B (UGT-B), rebaudioside D, and the same reaction system are as described above.

[0085] In one embodiment of the present application, the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, and further produces an isomer of rebaudioside D, but is not limited thereto.

[0086] Yet another aspect of the present application provides a method for producing rebaudioside M from rebaudioside A, the method comprising the step of producing rebaudioside M by reacting sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, glycosyltransferase A (UGT-A), and glycosyltransferase B (UGT-B) in a reaction system, wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3.

[0087] The sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, glycosyltransferase A (UGT-A), glycosyltransferase B (UGT-B), identical reaction system, and rebaudioside M are as described above.

[0088] Specifically, the glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, but is not limited to this.

[0089] In one embodiment of the present application, the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, and further produces an isomer of rebaudioside M, but is not limited thereto.

[0090] Yet another aspect of the present application provides a composition for producing rebaudioside D, comprising glycosyltransferase B (UGT-B), wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3.

[0091] The glycosyltransferase B (UGT-B) and rebaudioside D are as described above.

[0092] In one embodiment of the present application, the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the rebaudioside D is at least one selected from the group consisting of rebaudioside D and rebaudioside D isomers, but is not limited thereto.

[0093] Yet another aspect of the present application provides a composition for producing rebaudioside M, comprising glycosyltransferase A (UGT-A) and glycosyltransferase B (UGT-B), wherein the glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, and the glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequences set forth in SEQ ID NOs: 1 to 3.

[0094] The glycosyltransferase A (UGT-A), glycosyltransferase B (UGT-B), and rebaudioside M are as described above.

[0095] In one embodiment of the present application, the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the rebaudioside M is at least one selected from the group consisting of rebaudioside M and rebaudioside M isomers, but is not limited thereto.

[0096] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0097] Yet another aspect of the present application provides use of glycosyltransferase B (UGT-B), which is at least one protein selected from the group consisting of proteins having the amino acid sequences set forth in SEQ ID NOs: 1 to 3, as a glycosyltransferase that converts rebaudioside A to rebaudioside D.

[0098] The glycosyltransferase A (UGT-A), glycosyltransferase B (UGT-B), rebaudioside A, rebaudioside M, and the like are as described above. This application [Example]

[0099] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

[0100] Culture conditions A recombinant microorganism was prepared by introducing a polynucleotide encoding the glycosyltransferase B (UGT-B) of the present invention, and then the glycosyltransferase B (UGT-B) was expressed and used in the reaction. The culture conditions for the recombinant microorganism were as follows.

[0101] Example 1-1. Culture conditions for E. coli The recombinant E. coli was cultured by inoculating it into a test tube containing 5 ml of LB medium containing 50 μg / ml kanamycin, and culturing the inoculum in an incubator at 37°C until the absorbance at 600 nm reached 2.0. The culture medium containing the inoculum was added to a flask containing 500 ml of LB medium containing 50 μg / ml kanamycin, and main culturing was carried out. When the absorbance at 600 nm reached 0.4, 0.1 mM IPTG (isopropyl β-D-1-thiol) was added. The culture temperature was maintained at 37°C and the agitation speed was adjusted to 180 rpm. After IPTG addition, the agitation speed was increased to 120 rpm.

[0102] Example 1-2. Culture conditions for Corynebacterium The medium (Bacto-Trypton 10) contained kanamycin at a concentration of 10 μg / ml. g / L, Bacto-yeast extract 5g / L, NaCl 5g / L, So The recombinant Corynebacterium was added to the medium containing 5g / L of ytone at an initial concentration of OD600 = 0. The cells were inoculated at 1 mL and cultured at 30°C for 24 hours to induce enzyme expression. The cells were grown in a medium containing 10 μg / mL kanamycin (glucose 80 g / L, soytone 20 g / L, (NH 4) The culture solution obtained as described above was inoculated into a fermenter containing 10 g / L of KHSO, 1.2 g / L of KHPO, and 1.4 g / L of MgSO at an OD of 0.6, and cultured at 30°C for 24 hours. [Example]

[0103] Measurement of glycosyltransferase B (UGT-B) enzyme activity for rebaudioside A raw materials Example 2-1. Purification of glycosyltransferase B (UGT-B) The glycosyltransferase B (UGT-B; SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3) of the present application, which converts rebaudioside A to rebaudioside D, was prepared by constructing a recombinant plasmid (vector pET28a) containing a gene encoding the glycosyltransferase B of the present application (SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8), cloning the plasmid into Escherichia coli BL21(DE3), and then mass-expressing each enzyme, which was then purified and used.

[0104] Specifically, the recombinant strain BL21(DE3) was inoculated into a test tube containing 5 ml of LB medium, and the seed culture was cultured in an incubator at 37°C until the absorbance at 600 nm reached 2.0. The culture medium containing the seed culture was added to a flask containing 500 ml of LB medium, and main culture was carried out. When the absorbance at 600 nm reached 0.4, 0.1 mM IPTG was added. (isopropyl β-D-1-thiogalactopyranoside) was added to induce overexpression of the enzyme. During the above process, the stirring speed was adjusted to 180 rpm and the culture temperature to be maintained at 37°C. After the addition of IPTG, the stirring speed was adjusted to 120 rpm and the culture temperature to 16°C. The culture solution of the transformed strain was centrifuged at 6000 x g and 4°C for 20 minutes, and only the cell supernatant was separated as the enzyme solution. To accurately understand the properties of the enzyme, Ni-NTA Purification was carried out using a superflow column.

[0105] The enzymes overexpressed using recombinant plasmids containing SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 were designated UGT-B_6, UGT-B_7, and UGT-B_8, respectively.

[0106] On the other hand, a medium containing 10 μg / ml of kanamycin (Bacto-Trypton The recombinant Corynebacterium was added to a medium containing 10g / L of broth, 5g / L of yeast extract, 5g / L of NaCl, and 5g / L of soytone. The cells were inoculated at 0.1 and cultured at 30°C for 24 hours to induce enzyme expression. The cells were grown in a medium containing 10 μg / ml of kanamycin (glucose 80 g / L, soytone 20 g / L, ( NH4)2SO4 10g / L,KH2PO4 1.2g / L,MgSO4 1.4g / L The culture solution obtained as described above was inoculated into a fermenter containing 100 ml of HOAc at an OD600 of 0.6, and cultured at 30°C for 24 hours.

[0107] Example 2-2. Measurement of the enzyme activity of glycosyltransferase B (UGT-B) on rebaudioside A raw material Using each of the enzymes prepared in Example 2-1, the enzymatic activity of glycosyltransferase B (UGT-B) on rebaudioside A as a raw material was measured.

[0108] Specifically, the raw material used in the enzyme reaction was rebaudioside A (Daepyeong Co.), which was dissolved in water to a concentration of 2 mM. Using each enzyme expressed in the microorganisms prepared in Example 2-1, the reaction was carried out at 37°C for 16 hours, and then analyzed by HPLC. The aqueous solution of the raw material contained 2 mM of UDP-glucose or UDP (Uridine-diphosphate). That is why.

[0109] The HPLC analysis conditions were as follows. Detector wavelength: 210nm Flow rate: 1ml / min Sample injection volume: 10 μl ·Column:Capcell pak C18 MG II(Shiseido, 250×4 .6mm, particle size:5μm) Solvent: Acetonitrile 30%

[0110] The measurement results are shown in Table 1 and FIGS.

[0111] [Table 1]

[0112] As shown in Table 1 and Figures 1 to 3, it was confirmed that rebaudioside A was converted to rebaudioside D by glycosyltransferase B (UGT-B_6 and UGT-B_8) of the present application. It was confirmed that the glycosyltransferase (UGT-B_7) of the present application converts rebaudioside A to rebaudioside M isomers, rebaudioside D isomers, and rebaudioside D. [Example]

[0113] Measurement of glycosyltransferase A (UGT-A) and glycosyltransferase B (UGT-B) enzyme activities for stevioside raw materials Glycosyltransferase A (UGT-A) and sucrose synthase were purified according to the method described in the prior art document (Patent Document 2) and used. Their sequences are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

[0114] Using the glycosyltransferase A (UGT-A), the sucrose synthase, and each glycosyltransferase B (UGT-B) prepared in Example 2-1, the enzymatic activity of glycosyltransferase A (UGT-A) and glycosyltransferase B (UGT-B) on stevioside raw material was measured.

[0115] Specifically, the raw materials used in the enzyme reaction were an aqueous solution of 2 mM stevioside (Haigen) and 50 mM sugar (CJ CheilJedang). The glycosyltransferase A (UGT-A), the sucrose synthase, and each glycosyltransferase B (UGT-B) prepared in Example 2-1 were reacted at 37°C for 24 hours, and then analyzed by HPLC. The aqueous solution of raw materials contained 2 mM UDP-glucose or UDP (uridine-diphosphate). It includes.

[0116] The HPLC analysis conditions were as follows. Detector wavelength: 210nm Flow rate: 1ml / min Sample injection volume: 10 μl ·Column:Capcell pak C18 MG II(Shiseido, 250×4 .6mm, particle size:5μm) Solvent: Acetonitrile 30%

[0117] The measurement results are shown in Table 2 and FIGS.

[0118] [Table 2]

[0119] As shown in Table 2 and FIGS. 4 to 6, it was confirmed that stevioside was converted to rebaudioside M isomers, rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside A at a molar concentration of 100%. [Example]

[0120] Measurement of glycosyltransferase A (UGT-A) enzyme activity for rebaudioside D raw materials Using glycosyltransferase A (UGT-A) from Example 3, the conversion rate of rebaudioside D to rebaudioside M was measured.

[0121] Specifically, the raw material used in the enzyme reaction was rebaudioside D (Haigen) dissolved in water to a concentration of 1 mM. Using the glycosyltransferase A (UGT-A) expressed in a microorganism, the reaction was carried out at 37°C for 16 hours, and then analyzed by HPLC. The raw material aqueous solution contained 2 mM UDP-glucose or UDP (uridine-diphosphate). be.

[0122] The HPLC analysis conditions were as follows. Detector wavelength: 210nm Flow rate: 1ml / min Sample injection volume: 10 μl ·Column:Capcell pak C18 MG II(Shiseido, 250×4 .6mm, particle size:5μm) Solvent: Acetonitrile 30%

[0123] The measurement results are shown in Figure 7.

[0124] As shown in FIG. 7, it was confirmed that rebaudioside D was almost entirely converted to rebaudioside M by glycosyltransferase A of the present invention. [Example]

[0125] Analysis of Rebaudioside D Isomers and Rebaudioside M Isomers by Liquid Chromatography-Mass Spectrometry (LC-MS / MS) To confirm whether the compounds converted in Examples 2 and 3 were rebaudioside D isomers or rebaudioside M isomers, the rebaudioside D isomers and rebaudioside M isomers converted in Examples 2 and 3 were compared with rebaudioside D and rebaudioside M, respectively, using liquid chromatography mass spectrometry (LC-MS / MS) analysis.

[0126] Specifically, Acquity UPLC (Waters) and Xe-vo G2-XS Q- A Tof mass spectrometer was used. The LC-MS / MS analysis conditions are shown in Table 3, the elution conditions are shown in Table 4, and the MS conditions are shown in Table 5.

[0127] [Table 3]

[0128] [Table 4]

[0129] [Table 5]

[0130] As a result, as shown in Figures 8 and 9, the RebD Standard and fraction Comparison of the RebD standard and unknown peak 1 revealed that they have the same molecular weight and the same MSMS fragment, but the peak retention times are different, confirming that unknown peak 1 is an isomer of RebD.

[0131] In addition, as shown in Figures 10 and 11, the RebM Standard and fractio Comparison of the RebM standard and unknown peak 2 revealed that they have the same molecular weight and the same MSMS fragment, but the peak retention times are different, confirming that unknown peak 2 is an isomer of RebM.

[0132] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. reacting a glucose-linked nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B) to produce rebaudioside D; A method for producing rebaudioside D, wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences represented by SEQ ID NOs: 1 to 3.

2. The method according to claim 1 , wherein the glucose-linked nucleotide diphosphate is produced by reacting sucrose with a nucleotide diphosphate in the presence of sucrose synthase.

3. 2. The method according to claim 1, wherein the rebaudioside A is produced by reacting a glucose-linked nucleotide diphosphate with stevioside in the presence of glycosyltransferase A (UGT-A).

4. The method of claim 2 , wherein the sucrose synthase is a protein consisting of the amino acid sequence represented by SEQ ID NO:

5.

5. The method according to claim 3, wherein the glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

4.

6. 2. The method of claim 1, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 and further produces an isomer of rebaudioside D.

7. The method according to any one of claims 1 to 6, wherein the method is carried out continuously in the same reaction system.

8. reacting a glucose-linked nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B) to produce rebaudioside D; reacting the rebaudioside D with a glucose-linked nucleotide diphosphate in the presence of glycosyltransferase A (UGT-A) to produce rebaudioside M; A method for producing rebaudioside M, wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences represented by SEQ ID NOs: 1 to 3.

9. The method according to claim 8, wherein the glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

4.

10. The method according to claim 8 , wherein the glucose-linked nucleotide diphosphate is produced by reacting sucrose with a nucleotide diphosphate in the presence of sucrose synthase.

11. 9. The method according to claim 8, wherein the rebaudioside A is produced by reacting a glucose-linked nucleotide diphosphate with stevioside in the presence of glycosyltransferase A (UGT-A).

12. The method of claim 10, wherein the sucrose synthase is a protein consisting of the amino acid sequence represented by SEQ ID NO:

5.

13. The method according to claim 11, wherein the glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

4.

14. 9. The method of claim 8, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 and further produces an isomer of rebaudioside M.

15. The method according to any one of claims 8 to 14, wherein the method is carried out continuously in the same reaction system.

16. a step of reacting sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase, and glycosyltransferase B (UGT-B) in a single reaction system to produce rebaudioside D; The method for producing rebaudioside D from rebaudioside A, wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences represented by SEQ ID NOs: 1 to 3.

17. 17. The method for producing rebaudioside D from rebaudioside A according to claim 16, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 and further produces an isomer of rebaudioside D.

18. a step of reacting sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, glycosyltransferase A (UGT-A), and glycosyltransferase B (UGT-B) in a reaction system to produce rebaudioside M; The method for producing rebaudioside M from rebaudioside A, wherein the glycosyltransferase B is at least one protein selected from the group consisting of proteins having the amino acid sequences represented by SEQ ID NOs: 1 to 3.

19. 19. The method for producing rebaudioside M from rebaudioside A according to claim 18, wherein the glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence set forth in SEQ ID NO:

4.

20. 19. The method for producing rebaudioside M from rebaudioside A according to claim 18, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 and further produces an isomer of rebaudioside M.

21. A composition for producing rebaudioside D, comprising at least one glycosyltransferase B (UGT-B) selected from the group consisting of proteins having the amino acid sequences represented by SEQ ID NOs: 1 to 3.

22. 22. The composition for producing rebaudioside D according to claim 21, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, and the rebaudioside D is at least one selected from the group consisting of rebaudioside D and rebaudioside D isomers.

23. At least one glycosyltransferase B (UGT-B) selected from the group consisting of proteins having the amino acid sequences represented by SEQ ID NOs: 1 to 3; A composition for producing rebaudioside M, comprising glycosyltransferase A (UGT-A), a protein consisting of the amino acid sequence represented by SEQ ID NO:

4.

24. 24. The composition for producing rebaudioside M according to claim 23, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, and the rebaudioside M is at least one selected from the group consisting of rebaudioside M and rebaudioside M isomers.

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