Lactobacillus mali mutant strain having improved transglucosylation activity and method of preparing glucosylated steviol glycosides using same

Abstract

The present disclosure provides a mutant strain of Lactobacillus mali having improved steviol glycoside transglucosylation activity.

Description

Lactobacillus mali mutant strain with improved glucose transfer activity and method for producing glucose transfer steviol glycosides using the same

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0122517, filed September 9, 2024, and Korean Patent Application No. 10-2025-0119488, filed August 26, 2025, the entire contents of which are incorporated herein by reference.

[0003] [Technical Field]

[0004] The present disclosure relates to a mutant strain of Lactobacillus mali having improved glucose transfer activity, a method for producing a glucose transfer steviol glycoside using the mutant strain, and a sweetener composition comprising a steviol glycoside produced by the method.

[0005] As the World Health Organization (WHO) recommends reducing daily sugar intake due to concerns about obesity, type 2 diabetes, and cardiovascular disease, which are linked to excessive sugar consumption, policies to reduce sugar intake are being actively discussed, particularly in developed countries. Consequently, the market is seeing a rise in demand for various alternative sweeteners, particularly natural sweeteners that do not contain synthetic or chemical ingredients that can potentially negatively impact health.

[0006] Among the natural high-intensity sweeteners, stevioside and rebaudioside A are the most abundant steviol glycosides found in the leaves of Stevia rebaudiana Bertoni and are the most commonly used natural alternative sweeteners. However, they are still sensorially indistinguishable from sugar due to their bitterness, astringency, and long-lasting sweetness. Rebaudioside D and rebaudioside M, which exhibit good sweetness, are also found naturally in trace amounts, and their high price limits their application in food.

[0007] Enzymatically processed stevia has been studied to improve the limited taste and solubility of steviol glycosides, and is generally produced by glycosylating glucose into steviol glycosides via alpha-1,4 linkages using the enzyme cyclodextrin glucanotransferase (CGTase). However, as a more improved method, a Lactobacillus mali strain with the activity of alpha-1,6 linking 1 to 4 glucose molecules to rebaudioside A has been proposed, and the glucose transfer rebaudioside A produced using this has an improved sensory profile, such as bitterness, of rebaudioside A, and it was confirmed that the sensory profile, such as bitterness and astringency, is improved compared to enzymatically processed stevia with alpha-1,4 linkages.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] (Patent Document 1) U.S. Patent Publication No. US 2010-0189861

[0011] The purpose of the present disclosure is to provide a strain of Lactobacillus mali deposited with the Korean Culture Center of Microorganisms (KCCM) under the accession number KCCM 13503P.

[0012] Another object of the present disclosure is to provide a method for producing a glucose-transferred steviol glycoside, comprising the step of contacting the Lactobacillus mali strain, its culture solution or crude enzyme solution, with a glucose donor and a steviol glycoside.

[0013] Another object of the present disclosure is to provide a composition for producing glucose-transferred steviol glycosides comprising the Lactobacillus mali strain, a culture solution thereof, or a crude enzyme solution thereof.

[0014] Another object of the present disclosure is to provide a sweetening composition comprising a glucose-transferred steviol glycoside.

[0015] One aspect of the present disclosure provides a strain of Lactobacillus mali, deposited with the Korean Culture Center of Microorganisms (KCCM) under the deposit number KCCM13503P.

[0016] Another aspect of the present disclosure provides a method for producing a glucose-transferred steviol glycoside, comprising the step of contacting the Lactobacillus mali strain, a culture solution thereof, or a crude enzyme solution thereof with a glucose donor and a steviol glycoside.

[0017] Another aspect of the present disclosure provides a composition for producing glucose-transferred steviol glycosides comprising the Lactobacillus mali strain, a culture solution thereof, or a crude enzyme solution thereof.

[0018] Another aspect of the present disclosure provides a sweetening composition comprising a glucose-transferred steviol glycoside.

[0019] The present disclosure is described in more detail below.

[0020] According to one aspect of the present disclosure, there is provided a Lactobacillus mali strain deposited with the Korean Culture Center of Microorganisms (KCCM) under the deposit number KCCM 13503P.

[0021] The above Lactobacillus mali strain KCCM 13503P is a mutant strain obtained by inducing a mutation from Lactobacillus mali DSM20444.

[0022] In this specification, the Lactobacillus mali strain may be referred to interchangeably as “Lactobacillus mali KCCM13503P”, “Lactobacillus mali CJST242”, “KCCM13503P”, or “CJST242” strain names.

[0023] The Lactobacillus mali CJST242 strain of the present disclosure was deposited with the Korean Culture Center of Microorganisms, an international depository under the Budapest Treaty, on August 5, 2024, under the deposit number KCCM 13503P.

[0024] In one embodiment, the Lactobacillus mali KCCM13503P may comprise a DNA nucleotide sequence of SEQ ID NO: 1 encoding 16s rRNA.

[0025] The above Lactobacillus mali KCCM13503P may include a DNA nucleotide sequence including a mutated nucleotide compared to Lactobacillus mali DSM20444.

[0026] In one embodiment, the Lactobacillus mali KCCM13503P may include a DNA nucleotide sequence encoding a protein comprising an amino acid sequence of SEQ ID NO: 5; or a protein having an amino acid corresponding to position 156 of the sequence of SEQ ID NO: 5 that is asparagine and having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5.

[0027] In one embodiment, the Lactobacillus mali strain KCCM13503P may include a DNA nucleotide sequence encoding a protein comprising an amino acid sequence of SEQ ID NO: 6; or a protein having a threonine amino acid corresponding to position 196 of the sequence of SEQ ID NO: 6 and having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6.

[0028] In one embodiment, the Lactobacillus mali strain KCCM13503P may include a DNA nucleotide sequence encoding a protein comprising an amino acid sequence of SEQ ID NO: 7; or a protein having a leucine amino acid corresponding to position 127 of the sequence of SEQ ID NO: 7 and having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.

[0029] In one embodiment, the Lactobacillus mali KCCM13503P may comprise a DNA nucleotide sequence of SEQ ID NO: 2; or a DNA nucleotide sequence in which the nucleotide corresponding to position 466 of the sequence of SEQ ID NO: 2 has an adenine nucleotide and has sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with SEQ ID NO: 2.

[0030] In one embodiment, the Lactobacillus mali KCCM13503P may comprise a DNA nucleotide sequence of SEQ ID NO: 3; or a DNA nucleotide sequence having a cytosine nucleotide corresponding to the 587th nucleotide of the sequence of SEQ ID NO: 3 and having a sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with SEQ ID NO: 3.

[0031] In one embodiment, the Lactobacillus mali KCCM13503P may include a DNA nucleotide sequence of SEQ ID NO: 4, or a DNA nucleotide sequence in which the nucleotide corresponding to the 380th position of the sequence of SEQ ID NO: 4 has a thymine nucleotide and has a sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with SEQ ID NO: 4.

[0032] In one embodiment, sequence number 2 may be a DNA nucleotide sequence encoding a helicase C-terminal domain-containing protein.

[0033] In one embodiment, SEQ ID NO: 5 may be an amino acid sequence of a helicase C-terminal domain-containing protein.

[0034] In one embodiment, sequence number 3 may be a DNA nucleotide sequence encoding DNA polymerase III subunit alpha (dnaE).

[0035] In one embodiment, sequence number 6 may be the amino acid sequence of DNA polymerase III subunit alpha (dnaE).

[0036] In one embodiment, sequence number 4 may be a DNA nucleotide sequence encoding RNA polymerase recycling motor HelD (helD).

[0037] In one embodiment, sequence number 7 may be the amino acid sequence of RNA polymerase recycling motor HelD (helD).

[0038] In one embodiment, the Lactobacillus mali KCCM13503P may comprise a DNA nucleic acid molecule comprising at least one of the DNA nucleotide sequences described above.

[0039] In one embodiment, the Lactobacillus mali KCCM13503P may include at least one protein selected from the group consisting of (i) helicase C-terminal domain-containing protein, (ii) DNA polymerase III subunit alpha (dnaE), and (iii) RNA polymerase recycling motor HelD (helD), which comprises a mutated amino acid compared to the Lactobacillus mali DSM20444 described above.

[0040] According to another aspect of the present disclosure, a Lactobacillus mali strain having steviol glycoside glucose transfer activity is provided.

[0041] In one embodiment, the Lactobacillus mali strain may be a strain mutated from Lactobacillus mali DSM20444.

[0042] The above Lactobacillus mali strain may include a DNA nucleotide sequence included in the above-described Lactobacillus mali KCCM13503P.

[0043] The technical content regarding the DNA nucleotide sequence or amino acid sequence included in the above Lactobacillus mali strain is the same as that described in the above Lactobacillus mali KCCM13503P, and therefore is cited and not described again.

[0044] In one embodiment, the Lactobacillus mali may comprise a DNA nucleic acid molecule comprising at least one of the DNA nucleotide sequences described above.

[0045] In one embodiment, the Lactobacillus mali strain may comprise at least one protein selected from the group consisting of (i) a helicase C-terminal domain-containing protein, (ii) a DNA polymerase III subunit alpha (dnaE), and (iii) a RNA polymerase recycling motor HelD (helD), which comprises a mutated amino acid compared to the Lactobacillus mali DSM20444 described above.

[0046] The above Lactobacillus mali KCCM 13503P strain has glucose transfer activity.

[0047] In one embodiment, the glucose transfer activity comprises a steviol glycoside glucose transfer activity using a steviol glycoside as a substrate.

[0048] In the present disclosure, “steviol glycoside” is one of the natural sweeteners and is a substance represented by the following chemical formula 1.

[0049]

[0050] In the above chemical formula 1, R1 may have hydrogen (H) bonded to it, or 1 to 3 glucoses may be bonded to it via β bonds, and R2 may have 1 glucose, xylose, or rhamnose bonded to it via β bonds, and 0 to 2 glucoses may be bonded to it via α bonds, but is not limited thereto.

[0051] In the present disclosure, "transglucose steviol glycoside" refers to a compound to which one or more molecules of glucose are further added to the steviol glycoside. For example, when the steviol glycoside is rebaudioside A, transglucose steviol glycoside A refers to a compound to which one or more molecules of glucose are further added to rebaudioside A.

[0052] In one embodiment, the substrate steviol glycoside may include at least one selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, stevioside, dulcoside A, steviolbioside, and rubusoside.

[0053] In one embodiment, the Lactobacillus mali strain of the present disclosure described above may have improved steviol glycoside glucose transfer activity compared to Lactobacillus mali DSM20444.

[0054] The improvement in the glucose transfer activity of the steviol glycoside may be due to an increase in the glucose transfer activity or an increase in the amount of the glucose transfer activity enzyme.

[0055] Since the Lactobacillus mali KCCM 13503P of the present disclosure has improved glucose transfer activity, it can efficiently produce a high content of glucose-transferred steviol glycosides even with a small amount of strain culture solution or strain crude enzyme solution, and can shorten the reaction time of the steviol glycoside glucose transfer reaction.

[0056] In one embodiment, the Lactobacillus mali KCCM 13503P strain may have a steviol glycoside glucose transfer activity increased by 1.4 times or more compared to the Lactobacillus mali DSM20444 strain, specifically, it may be 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2 times or more, and more specifically, 1.4 - 4 times, 1.4 - 3.5 times, 1.4 - 3 times, 1.4 - 2.7 times, 1.4 - 2.5 times, 1.4 - 2.4 times, 1.4 - 2.3 times, 1.4 - 2.2 times, 1.4 - 2.1 times, 1.5 - 4 times, 1.5 - 3.5 times, 1.5 - It can be, but is not limited to, 3x, 1.5 - 2.7x, 1.5 - 2.5x, 1.5 - 2.4x, 1.5 - 2.3x, 1.5 - 2.2x, 1.5 - 2.1x, 1.7 - 4x, 1.7 - 3.5x, 1.7 - 3x, 1.7 - 2.7x, 1.7 - 2.5x, 1.7 - 2.4x, 1.7 - 2.3x, 1.7 - 2.2x, 1.7 - 2.1x.

[0057] In one embodiment, the conversion rate of rebaudioside A of the Lactobacillus mali KCCM 13503P into glucose transfer rebaudioside A may be 1.4 times or more compared to the Lactobacillus mali DSM20444 strain, and specifically, may be 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2 times or more compared to the Lactobacillus mali DSM20444. More specifically, the conversion rate of the mutant strain KCCM of Lactobacillus mali is 1.4 - 4 times, 1.4 - 3.5 times, 1.4 - 3 times, 1.4 - 2.7 times, 1.4 - 2.5 times, 1.4 - 2.4 times, 1.4 - 2.3 times, 1.4 - 2.2 times, 1.4 - 2.1 times, 1.5 - 4 times, 1.5 - 3.5 times, 1.5 - 3 times, 1.5 - 2.7 times, 1.5 - 2.5 times, 1.5 - 2.4 times, 1.5 - 2.3 times, 1.5 - 2.2 times, 1.5 - 2.1 times, 1.7 - 4 times, 1.7 - It can be, but is not limited to, 3.5x, 1.7 - 3x, 1.7 - 2.7x, 1.7 - 2.5x, 1.7 - 2.4x, 1.7 - 2.3x, 1.7 - 2.2x, 1.7 - 2.1x.

[0058] In one embodiment, the conversion rate may be the conversion rate of rebaudioside A to glucose transfer rebaudioside A measured in a reaction product comprising 10 wt% of a Lactobacillus mali strain culture, 10 wt% of sugar, and 10 wt% of rebaudioside A, reacted at 35 to 45°C for 0.5 to 2 hours.

[0059] In one embodiment, the improvement in the glucose transfer activity of the steviol glycoside may comprise an increase in the number of glucose molecules transferred to the steviol glycoside.

[0060] In one embodiment, the Lactobacillus mali KCCM13503P may have an activity of transferring 1 to 11 glucose molecules to a steviol glycoside, and specifically, the number of glucose molecules transferred may be 1 to 11 molecules, 2 to 11 molecules, 3 to 11 molecules, 4 to 11 molecules, 5 to 11 molecules, 6 to 11 molecules, 7 to 11 molecules, 8 to 11 molecules, 9 to 11 molecules, 10 to 11 molecules, or 11 molecules.

[0061] In one embodiment, the steviol glycoside to which 1 to 11 glucose molecules are transferred may include at least one selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, stevioside, dulcoside A, steviolbioside, and rubusoside.

[0062] In one embodiment, the steviol glycoside to which the 1 to 11 glucose molecules are transferred may comprise at least one selected from the group consisting of rebaudioside A, rebaudioside C, rebaudioside F, stevioside, dulcoside A, and rubusoside.

[0063] In one embodiment, the transfer of 1 to 11 glucose molecules to a steviol glycoside by the Lactobacillus mali KCCM13503P comprises transfer of 1 to 11 glucose molecules via an α-(1,6) linkage to a glucose molecule linked to 19-OH of the steviol glycoside.

[0064] According to another aspect of the present disclosure, a bacterial composition comprising the above-described Lactobacillus mali KCCM13503P or Lactobacillus mali strain is provided.

[0065] In one embodiment, the composition may further comprise a cryoprotectant.

[0066] The Lactobacillus mali KCCM13503P or Lactobacillus mali strain of the present disclosure can be prepared in the form of a lyophilized composition to preserve the activity of the microbial cells, and the composition can further include a cryoprotectant to prevent damage to the cells.

[0067] In one embodiment, the cryoprotectant may include one or more selected from the group consisting of glycerol, ethylene glycol, propylene glycol, trehalose, sucrose, glucose, maltodextrin, sorbitol, skimmed milk powder, and starch.

[0068] In one embodiment, the cryoprotectant may be present in an amount of 0.01 to 30 wt% relative to the total composition, specifically 0.01 to 30 wt%, 0.01 to 25 wt%, 0.01 to 20 wt%, 0.01 to 15 wt%, 0.1 to 30 wt%, 0.1 to 25 wt%, 0.1 to 20 wt%, 0.1 to 15 wt%, 1 to 30 wt%, 1 to 25 wt%, 1 to 20 wt%, or 1 to 15 wt% relative to the total composition.

[0069] In one embodiment, the Lactobacillus mali KCCM13503P is 10% of the total composition. 4 It may contain more than cfu / ml, specifically 10 4 10 inland 14 cfu / ml, 10 4 10 inland 13 cfu / ml, 10 4 10 inland 12 cfu / ml, 10 5 10 inland 14 cfu / ml, 10 5 10 inland 13 cfu / ml, 10 5 10 inland 12 cfu / ml, 10 610 inland 14 cfu / ml, 10 6 10 inland 13 cfu / ml, 10 6 10 inland 12 cfu / ml, 10 4 10 inland 14 cfu / ml, or 10 4 10 inland 14 It can be included in the amount of cfu / ml.

[0070] In one embodiment, the bacterial composition may be in a lyophilized form.

[0071] In one embodiment, the bacterial composition may be in the form of a lyophilized powder.

[0072] According to another aspect of the present disclosure, a method for producing a glucose-transferred steviol glycoside is provided, comprising the step of contacting the above-described Lactobacillus mali KCCM13503P strain, a culture solution or a crude enzyme solution of the strain, with a glucose donor and a steviol glycoside.

[0073] In the present disclosure, the "enzyme solution" may include a fraction containing glucose transferase, and may include, for example, a supernatant of a culture of Lactobacillus mali KCCM13503P strain.

[0074] In the present disclosure, the "glucose donor" may be any of oligomers of glucose, polymers of glucose and cyclic forms thereof, which can be reacted in the presence of a glucose transferase to transfer one or more glucoses to steviol glycosides.

[0075] In one embodiment, the glucose donor may be a “disaccharide comprising glucose,” wherein the disaccharide comprising glucose means a disaccharide comprising at least glucose as a monosaccharide constituting the disaccharide.

[0076] In one embodiment, the “disaccharide comprising glucose” comprises sucrose, lactose, or maltose.

[0077] In one embodiment, the glucose donor may be sucrose or sugar.

[0078] In one embodiment, the steviol glycoside may include at least one selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, stevioside, dulcoside A, steviolbioside, and rubusoside.

[0079] In the present disclosure, by contacting the Lactobacillus mali KCCM13503P strain, the culture solution or the crude enzyme solution of the strain with a glucose donor and a steviol glycoside, the glucose transfer reaction of the steviol glycoside progresses, thereby producing a glucose transfer steviol glycoside.

[0080] In one embodiment, the glucose transfer reaction of the steviol glycoside can be performed in a reaction solution containing the Lactobacillus mali KCCM13503P strain, a culture solution or a crude enzyme solution of the strain, a glucose donor, and a steviol glycoside.

[0081] In one embodiment, the culture solution or crude enzyme solution of the Lactobacillus mali KCCM13503P strain may be included in an amount of 1 to 40 wt%, 1 to 35 wt%, 1 to 30 wt%, 3 to 40 wt%, 3 to 35 wt%, 3 to 30 wt%, 5 to 40 wt%, 5 to 35 wt%, 5 to 30 wt%, 7 to 40 wt%, 7 to 35 wt%, 7 to 30 wt%, 10 to 40 wt%, 10 to 35 wt%, or 10 to 30 wt% relative to the total reaction solution.

[0082] In one embodiment, the glucose donor may be included in an amount of 1 to 20 wt%, 1 to 17 wt%, 1 to 15 wt%, 3 to 20 wt%, 3 to 17 wt%, 3 to 15 wt%, 5 to 20 wt%, 5 to 17 wt%, or 5 to 15 wt% relative to the total reaction solution.

[0083] In one embodiment, the steviol glycoside may be included in an amount of 1 to 20 wt%, 1 to 17 wt%, 1 to 15 wt%, 3 to 20 wt%, 3 to 17 wt%, 3 to 15 wt%, 5 to 20 wt%, 5 to 17 wt%, or 5 to 15 wt% relative to the total reaction solution.

[0084] In one embodiment, the “glucose donor” and the “steviol glycoside” in the glucose transfer reaction solution may be included in a weight ratio of glucose donor: steviol glycoside = 1:2 to 2:1.

[0085] In one embodiment, the glucose transfer reaction of the steviol glycoside can be performed at a temperature of 20 to 50°C, 25 to 50°C, 30 to 50°C, 35 to 50°C, 30 to 45°C, or 35 to 45°C.

[0086] In one embodiment, the glucose transfer reaction of the steviol glycoside can be performed in the range of 0.1 to 10 hours, 0.1 to 8 hours, 0.1 to 7 hours, 0.1 to 6 hours, 0.2 to 5 hours, 0.3 to 5 hours, 0.5 to 5 hours, or 1 to 5 hours.

[0087] According to another aspect of the present disclosure, a composition for producing glucose-transferred steviol glycosides is provided, comprising the Lactobacillus mali KCCM13503P strain described above, a culture solution or a crude enzyme solution of the strain.

[0088] In one embodiment, the composition for producing steviol glycosides may further comprise a glucose donor and a steviol glycoside.

[0089] In the present disclosure, the "glucose donor" may be any of oligomers of glucose, polymers of glucose and cyclic forms thereof, which can be reacted in the presence of a glucose transferase to transfer one or more glucoses to steviol glycosides.

[0090] In one embodiment, the glucose donor may be a “disaccharide comprising glucose,” wherein the disaccharide comprising glucose means a disaccharide comprising at least glucose as a monosaccharide constituting the disaccharide.

[0091] In one embodiment, the “disaccharide comprising glucose” comprises sucrose, lactose, or maltose.

[0092] In one embodiment, the glucose donor may be sucrose or sugar.

[0093] In one embodiment, the steviol glycoside may include at least one selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, stevioside, dulcoside A, steviolbioside, and rubusoside.

[0094] In one embodiment, the culture solution or crude enzyme solution of the Lactobacillus mali KCCM13503P strain may be included in an amount of 1 to 40 wt%, 1 to 35 wt%, 1 to 30 wt%, 3 to 40 wt%, 3 to 35 wt%, 3 to 30 wt%, 5 to 40 wt%, 5 to 35 wt%, 5 to 30 wt%, 7 to 40 wt%, 7 to 35 wt%, 7 to 30 wt%, 10 to 40 wt%, 10 to 35 wt%, or 10 to 30 wt% relative to the total composition.

[0095] In one embodiment, the glucose donor can be included in an amount of 1 to 20 wt%, 1 to 17 wt%, 1 to 15 wt%, 3 to 20 wt%, 3 to 17 wt%, 3 to 15 wt%, 5 to 20 wt%, 5 to 17 wt%, or 5 to 15 wt% relative to the total composition.

[0096] In one embodiment, the steviol glycoside may be included in an amount of 1 to 20 wt%, 1 to 17 wt%, 1 to 15 wt%, 3 to 20 wt%, 3 to 17 wt%, 3 to 15 wt%, 5 to 20 wt%, 5 to 17 wt%, or 5 to 15 wt% relative to the total composition.

[0097] In one embodiment, the “glucose donor” and the “steviol glycoside” in the composition may be included in a weight ratio of glucose donor: steviol glycoside = 1:2 to 2:1.

[0098] According to another aspect of the present disclosure, a sweetening composition comprising a glucose-transferred steviol glycoside is provided.

[0099] The above steviol glycoside may include at least one selected from the group consisting of rebaudioside A, rebaudioside C, rebaudioside F, stevioside, dulcoside A, and rubusoside.

[0100] The above glucose-transferred steviol glycoside may comprise 1 to 11 transferred glucose molecules.

[0101] In one embodiment, the glucose trans-steviol glycoside can comprise 1 to 11 trans-glucose molecules, specifically, the glucose trans-steviol glycoside can comprise 1 to 11, 2 to 11, 3 to 11, 4 to 11, 5 to 11, 6 to 11, 7 to 11, 8 to 11, 9 to 11, 10 to 11, or 11 trans-glucose molecules.

[0102] In one embodiment, the glucose transferred in the glucose transfer steviol glycoside may be added by linking it to the glucose linked to the 19-OH position of the steviol glycoside by an α-(1,6) bond.

[0103] In one embodiment, the glucose trans-steviol glycoside may be produced by a method for producing a glucose trans-steviol glycoside, which comprises a step of contacting the Lactobacillus mali KCCM13503P strain, a culture solution or a crude enzyme solution of the strain, with a glucose donor and a steviol glycoside.

[0104] The present disclosure provides Lactobacillus mali KCCM13503P (CJST242 strain) having improved glucose transfer activity.

[0105] The above Lactobacillus mali KCCM13503P strain has improved steviol glycoside glucose transfer activity, and can produce a high content of glucose-transferred steviol glycoside even with a small amount of strain culture medium, and can significantly shorten the reaction time of the glucose transfer reaction. In addition, the Lactobacillus mali KCCM13503P strain can significantly increase the number of glucose molecules transferred to steviol glycosides, and can significantly improve the sweetness of glucose-transferred steviol glycosides.

[0106] However, the effects of the present application are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0107] Figure 1 is a graph comparing the conversion rate of rebaudioside A to glucose transfer rebaudioside A over time in the parent strain Lactobacillus mali DSM20444 and Lactobacillus mali CJST242, which was obtained through mutation therefrom.

[0108] Figures 2a and 2b are graphs comparing the growth rates of seed growth and main growth of the parent strain Lactobacillus mali DSM20444 and Lactobacillus mali CJST242 obtained through mutation therefrom.

[0109] Figure 3a is an HPLC chromatogram of the result of the glycosylation reaction of rebaudioside A by Lactobacillus mali CJST242 strain.

[0110] Figure 3b is an HPLC chromatogram of the result of the glycosylation reaction of rebaudioside C by Lactobacillus mali CJST242 strain.

[0111] Figure 3c is an HPLC chromatogram of the result of the glycosylation reaction of rebaudioside F by Lactobacillus mali CJST242 strain.

[0112] Figure 3d is an HPLC chromatogram of the results of the transglycosylation reaction of stevioside by Lactobacillus mali CJST242 strain.

[0113] Figure 3e is an HPLC chromatogram of the result of the glycosylation reaction of dulcoside by Lactobacillus mali CJST242 strain.

[0114] Figure 3f is an HPLC chromatogram of the result of the glycosylation reaction of rubusoside by Lactobacillus mali CJST242 strain.

[0115] Hereinafter, the present application will be described in detail by way of examples. However, the following examples are intended to specifically illustrate the present application, and the contents of the present application are not limited by the following examples.

[0116] Example 1: Preparation of a mutant strain of Lactobacillus mali by irradiation

[0117] Lactobacillus mali DSM20444 strain (parent strain) provided by the Korea Center for Microorganism Conservation (KCCM) was cultured in MRS broth medium until log phase, and the cultured culture was irradiated (Advanced Radiation Research Institute, Korea Atomic Energy Research Institute). To determine the appropriate radiation dose, the radiation dose was varied to 0, 1, 1.5, 2, 2.5, and 3 kGy / hr, and the irradiated culture medium was spread on a solid medium to measure the death rate. Table 1 shows the results of comparing the death rate of Lactobacillus mali DSM20444 according to the radiation dose. From the death rate measurement results in Table 1, 1.5 kGy / hr, which is the highest dose among those with a death rate less than 100, was determined to be the appropriate dose to induce mutations.

[0118] Radiation dose DSM20444 strain dilution rate CFU / mL mortality rate (%) 10 0 10 -1 10 -2 10 -3 10 -4 10 -5 0 kGy / hrLawnLawnLawnLawn973838383,838,000,000-1 kGy / hr129362898542101171776,00099.979781.5 kGy / hr614122120009,17099.999762 kGy / hr200000201002.5 kGy / hr000000-1003 kGy / hr000000-100

[0119] The parent strain culture was irradiated at a dose of 1.5 kGy / hr, and the irradiated culture was spread on a solid medium and cultured at 30°C for 3 to 5 days to obtain a pool of strains containing the Lactobacillus mali strains that had undergone mutations. Each colony of the candidate strains for mutations was selected from the strain pool and cultured, and the conversion rate of the culture medium of each colony (strain) to convert rebaudioside A into glycosylated rebaudioside A was measured. Based on the results of the conversion rate measurement, strains with a better conversion rate than the parent strain were selected, and the mutant strain with the best conversion rate among the selected strains was finally selected. The mutant strain of Lactobacillus mali with the best conversion rate was named Lactobacillus mali CJST242. In order to compare the conversion rates of the parent strain and the mutant strain, the parent strain (DSM20444) and the mutant strain (CJST242) were cultured in MRS broth Modified medium to obtain a culture medium, and then sugar (CJ CheilJedang) and rebaudioside A (Daepyung) were added to the culture medium and a sugar conversion reaction was performed. The conversion rate of rebaudioside A to the sugar conversion was measured. The conversion rate was calculated by analyzing the decrease in rebaudioside A over time using HPLC. The results of the conversion rate measurement over time of the parent strain and the mutant strain measured under the same reaction conditions are shown in Fig. 1. From the results of Fig. 1, it was confirmed that the mutant strain Lactobacillus mali CJST242 had a conversion rate about 1.48 times higher than that of the parent strain Lactobacillus mali DSM20444 at a reaction time of 24 hours.

[0120] Example 2: Measurement of growth rate of Lactobacillus mali mutant strains

[0121] To compare the growth rates of the parent strain Lactobacillus mali DSM20444 and the mutant strain Lactobacillus mali CJST242, each strain was inoculated into 10 mL of MRS Broth medium and cultured in a 30℃ incubator. In addition, each strain was inoculated with 1-5% of the seed culture in 10 mL of MRS Broth Modified medium and cultured in a 30℃ incubator. The absorbance of each culture was measured at 600 nm over time, and the growth curves for seed growth and main growth are shown in Fig. 2. As shown in Fig. 2, in seed growth, the parent strain and the mutant strain reached the stationary phase at the same time, and the mutant strain showed a higher growth rate until the stationary phase. In main growth, the parent strain and mutant strain reached the stationary phase at the same time, and the mutant strain showed a higher growth rate than the parent strain.

[0122] Example 3: Identification of 16s rRNA nucleotide sequences and mutated DNA nucleotide sequences in the genome of Lactobacillus mali mutant strains.

[0123] In order to identify Lactobacillus mali CJST242 selected in the above Example 1, the DNA nucleotide sequence of the 16s rRNA gene was analyzed, and the sequence is shown in SEQ ID NO: 1. As a result of the analysis, the DNA sequence of the 16s rRNA gene of Lactobacillus mali CJST242 was identical to that of the parent strain, and thus, it was confirmed that the strain was the same Lactobacillus mali strain as the parent strain. In addition, as a result of analyzing the DNA sequences of genes essential for microbial growth for the Lactobacillus mali CJST242 strain, it was confirmed that the DNA sequences of three genes were different from those of the parent strain, as shown in Table 2 below (Table 2).

[0124] NumberProtein NameAmino Acid MutationDNA Nucleotide Mutation1Helicase C-terminal domain-containing proteinAsp156AsnSEQ ID NO: 5466G>ASEQ ID NO: 22DNA Polymerase III subunit alpha (dnaE)Lys196ThrSEQ ID NO: 6587A>CSEQ ID NO: 33RNA polymerase recycling motor HelD (helD)Arg127LeuSEQ ID NO: 7380G>TSEQ ID NO: 4

[0125] Lactobacillus mali CJST242, which was selected in Example 1 and had excellent efficiency in producing steviol glycosides, was confirmed to be a mutant strain with high homology to Lactobacillus mali based on the analysis of the DNA sequence encoding 16s rRNA, the DNA sequence of the mutated gene, growth rate, and enzyme productivity. This mutant strain was named Lactobacillus mali CJST242 and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository under the Budapest Treaty, on August 5, 2024, and was assigned the accession number KCCM13503P.

[0126] Example 4: Comparative evaluation of glucose transfer rebaudioside A production capacity of parent strain and mutant strain

[0127] Both the parent strain Lactobacillus mali DSM20444 and Lactobacillus mali CJST242 selected in Example 1 were OD 600 = The culture medium for each strain was prepared by culturing until it reached 1.6. The culture medium of the parent strain or mutant strain was added to the sugar transfer reaction medium to make 10 wt% or 30 wt%, and 10 wt% of sugar (CJ CheilJedang) and 10 wt% of rebaudioside A (Daepyung) were further added to prepare the reaction medium. The sugar transfer reaction was performed in the reaction medium in a constant temperature water bath at 40℃ while varying the reaction time in the range of 0.5 to 3 hours. After completion of the reaction, the amount of rebaudioside A decreased in each reaction medium was confirmed through HPLC analysis, and from the HPLC analysis results, the conversion rate of the sugar transfer reaction according to the amount of culture medium (enzyme) added and the reaction time was calculated for the parent strain and mutant strain. The calculated conversion rates are compared and shown in Table 3.

[0128] StrainMother strain (DSM20444)Mutant strain (CJST242)Conversion rate ratio (mutant strain / mother strain)Culture amount added 10%30%10%30%10%30%Reaction time 0.5h19.845.441.679.22.11.741h33.268.172.393.32.11.372h52.787.790.195.81.71.093h65.593.995.394.71.41.0

[0129] (In Table 3 above, the % of culture medium added is weight %, and the conversion rate unit is %) As a result of confirming the conversion rate of rebaudioside A of the parent strain and the mutant strain according to the amount of culture medium (enzyme) added and the reaction time, at 10 wt% of culture medium (enzyme) added and 0.5 - 3 hours of sugar transfer reaction, the parent strain showed a conversion rate of 19.8 - 65.5%, and the mutant strain showed a conversion rate of 41.6 - 95.3%, showing that the conversion rate of the mutant strain was superior. In addition, at a 30 wt% culture medium (enzyme) addition amount and a 0.5 to 2 hour sugar transfer reaction, the parent strain showed a conversion rate of 45.4 to 87.7%, and the mutant strain showed a conversion rate of 79.2 to 95.8%, and at a 30 wt% culture medium (enzyme) addition amount and a 3 hour reaction, the parent strain showed a conversion rate of 93.9%, and the mutant strain showed a conversion rate of 94.7%.

[0130] From this, it was confirmed that the conversion rate of the mutant strain was much superior to that of the parent strain in the early stage of the reaction, 0.5 to 2 hours. In addition, the parent strain achieved a conversion rate of over 90% after 3 hours of reaction at a 30% culture medium (enzyme) addition amount, whereas the mutant strain achieved a conversion rate of over 90% after 2 hours of reaction even at a 10% culture medium (enzyme) addition amount. From these results, the Lactobacillus mali CJST242 strain was able to achieve a conversion rate equivalent to that of the parent strain in a shorter time even at a lower culture medium addition amount, and reached a conversion rate equivalent to that of the parent strain in a significantly shorter time at the same culture medium addition amount.

[0131] Example 5: Evaluation of Glucose Transfer Rebaudioside A Production Ability from High-Concentration Rebaudioside A of Mutant Strain

[0132] A reaction solution was prepared by adding 10 wt%, 20 wt%, or 30 wt% of Lactobacillus mali CJST242 culture medium to a sugar transfer reaction solution, further adding 15 wt%, 20 wt%, or 30 wt% of sugar (CJ CheilJedang), and 15 wt%, 20 wt%, or 30 wt% of rebaudioside A (Daepyung). The sugar transfer reaction was performed in a constant temperature water bath at 40°C while varying the reaction time in the range of 1 to 9 hours. After completion of the reaction, the amount of rebaudioside A decreased in each reaction solution was confirmed through HPLC analysis, and the conversion rate of the sugar transfer reaction was calculated according to the amount of culture medium added, the amount of reaction raw material added, and the reaction time from the HPLC analysis results. The calculated conversion rates are shown in Table 4.

[0133] Enzyme (culture medium) 10% 20% 30% RebA 15% 20% 30% 15% 20% 30% 15% 20% 30% Sucrose 15% 20% 30% 15% 20% 30% 15% 20% 30% Reaction time 1h 36.326.313.861.344.821.484.468.841.49h 94.189.264.993.089.177.287.884.171.9

[0134] (In Table 4 above, the % of enzyme, RebA, and Sucrose are weight %, and the conversion rate unit is %) As a result of confirming the conversion rate of glucose to Rebaudioside A according to the amount of mutant strain culture added, the amount of reaction raw material added, and the reaction time, it was confirmed that in the reaction solution with 10 wt% of mutant strain culture added, when 15 wt%, 20 wt%, and 30 wt% of Rebaudioside A were added, the maximum conversion rates were 94.1%, 89.2%, and 64.9%, respectively. In addition, it was confirmed that as the amount of culture solution (enzyme) added increased from 10 wt% to 30 wt%, the initial reaction rate at a reaction time of 1 hour increased in proportion to the amount of culture solution added. Through these experimental results, it was confirmed that by adding 10-30 wt% of the culture solution of Lactobacillus mali CJST242 and reacting with 15-20 wt% of high-concentration rebaudioside A for 9 hours, at least 84.1% of glucose can be converted to rebaudioside A.

[0135] Example 6: Evaluation of glucose transfer ability of steviol glycosides of mutant strains

[0136] 10 wt% of Lactobacillus mali CJST242 culture was added to a sugar transfer reaction solution, 5 wt% of sugar (CJ CheilJedang), and 2 wt% of steviol glycosides such as rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, stevioside, dulcoside A, steviolbioside, or rubusoside were further added to prepare sugar transfer reaction solutions. The prepared reaction solutions were reacted in a constant temperature water bath at 40°C to perform a sugar transfer reaction.

[0137] After the reaction was completed, HPLC analysis was performed on each reaction solution to obtain HPLC chromatograms. Each HPLC chromatogram according to the type of steviol glycoside is shown in Figs. 3a, 3b, 3c, 3d, 3e, and 3f. Specifically, Fig. 3a is an HPLC chromatogram of the result of the glycosyltransfer reaction of rebaudioside A, Fig. 3b is an HPLC chromatogram of the result of the glycosyltransfer reaction of rebaudioside C, Fig. 3c is an HPLC chromatogram of the result of the glycosyltransfer reaction of rebaudioside F, Fig. 3d is an HPLC chromatogram of the result of the glycosyltransfer reaction of stevioside, Fig. 3e is an HPLC chromatogram of the result of the glycosyltransfer reaction of dulcoside, and Fig. 3f is an HPLC chromatogram of the result of the glycosyltransfer reaction of rubusoside.

[0138] From the results of FIGS. 3a to 3f, it was confirmed that in the sugar transfer reaction solution prepared using the culture medium of Lactobacillus mali CJST242, rebaudioside A transferred 1 to 11 glucose units, rebaudioside C transferred 1 to 11 glucose units, rebaudioside F transferred 1 to 11 glucose units, stevioside transferred 1 to 11 glucose units, dulcoside A transferred 1 to 11 glucose units, and rubusoside transferred 1 to 11 glucose units.

[0139] Additionally, the results of LC-MS / MS analysis of each peak in each HPLC chromatogram according to the type of steviol glycoside are shown in Tables 5 to 10.

[0140] Table 5 below shows the results of LC-MS / MS analysis of the glycosylation reaction solution of rebaudioside A using Lactobacillus mali CJST242 culture and the results of predicting the chemical structure of the product.

[0141] Table 6 below shows the results of LC-MS / MS analysis of the glycosylation reaction solution of rebaudioside C using Lactobacillus mali CJST242 culture and the results of predicting the chemical structure of the product.

[0142] Table 7 below shows the results of LC-MS / MS analysis of the glycosylation reaction solution of rebaudioside F using Lactobacillus mali CJST242 culture and the results of predicting the chemical structure of the product.

[0143] Table 8 below shows the results of LC-MS / MS analysis of the sugar transfer reaction solution of stevioside using Lactobacillus mali CJST242 culture and the results of predicting the chemical structure of the product.

[0144] Table 9 below shows the results of LC-MS / MS analysis of the glycosylation reaction solution of dulcoside A using Lactobacillus mali CJST242 culture and the results of predicting the chemical structure of the product.

[0145] Table 10 below shows the results of LC-MS / MS analysis of the glycosylation reaction solution of rubusoside using Lactobacillus mali CJST242 culture and the results of predicting the chemical structure of the product.

[0146] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification17.93965.4214[MH] - 965.4230-1.6C44H70O23966.4308317, 479, 641, 803, 965Rebaudioside A29.931127.4803[MH] - 1127.47584.0C50H80O281128.4836317, 479, 641, 803, 1127Reb A + 1 Glucose311.311289.5314[MH] - 1289.52862.2C56H90O331290.5364317, 479, 641, 803, 1289Reb A + 2 Glucose412.281451.5854[MH] -1451.58142.7C62H100O381452.5893317, 479, 641, 803, 1451Reb A + 3 Glucose513.091613.6410[M-H] - 1613.63434.2C68H110O431614.6421317, 479, 641, 803, 809, 1613Reb A + 4 Glucose613.761775.6942[M-H] - 1775.68714.0C74H120O481776.6949317, 479, 641, 803, 971, 1775Reb A + 5 Glucose714.36968.3641[M-2H] 2- 968.3660-2.0C80H130O531938.7477317, 479, 641, 803, 968, 1133Reb A + 6 Glucose814.891049.3915[M-2H] 2- 1049.3925-0.9C86H140O582100.8006317, 479, 641, 803, 1049, 1295Reb A + 7 Glucose915.371130.4181[M-2H] 2- 1130.4189-0.7C92H150O632262.8534317, 479, 641, 803, 1130, 1457Reb A + 8 Glucose1015.721211.4421[M-2H] 2- 1211.4453-2.6C98H160O682424.9062317, 479, 641, 803, 1211, 1619Reb A + 9 Glucose1116.041292.4698[M-2H] 2- 1292.4717-1.5C104H170O732586.9590317, 479, 641, 803, 1292, 1781Reb A + 10 Glucose1216.331373.5012[M-2H] 2- 1373.49812.2C110H180O782749.0119317, 479, 641, 803, 1373Reb A + 11 Glucose

[0147] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification16.85949.4280[M-H] - 949.4281-0.1C44H70O22950.4359317, 479, 625, 787, 949Rebaudioside C29.221111.4812[M-H] - 1111.48090.3C50H80O271112.4887317, 479, 625, 787, 1111Reb C + 1 Glucose310.661273.5367[M-H] - 1273.53372.4C56H90O321274.5415317, 479, 625, 787, 1273Reb C + 2 Glucose411.821435.5806[M-H] - 1435.5865-4.1C62H100O371436.5944317, 479, 625, 787, 1435Reb C + 3 Glucose512.311597.6388[M-H] - 1597.6394-0.3C68H110O421598.6472317, 479, 625, 787, 809, 1597Reb C + 4 Glucose613.341759.6941[M-H] - 1759.69221.1C74H120O471760.7000317, 479, 625, 787, 971, 1759Reb C + 5 Glucose713.97960.3698[M-2H] 2- 960.36861.3C80H130O521922.7528317, 479, 625, 787, 960, 1133Reb C + 6 Glucose814.531041.3955[M-2H] 2-1041.39500.5C86H140O572084.8057317, 479, 625, 787, 1041, 1295Reb C + 7 Glucose915.021122.4226[M-2H] 2- 1122.42141.1C92H150O622246.8585317, 479, 625, 787, 1122, 1457Reb C + 8 Glucose1015.461203.4496[M-2H] 2- 1203.44781.5C98H160O672408.9113317, 479, 625, 787, 1203, 1619Reb C + 9 Glucose1115.821284.4769[M-2H] 2- 1284.47422.1C104H170O722570.9641317, 479, 625, 787, 1284, 1781Reb C + 10 Glucose1216.201365.5046[M-2H] 2- 1365.50072.9C110H180O772733.0170317, 479, 625, 787, 1365, 1943Reb C + 11 Glucose

[0148] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification17.12935.4150[M-H] - 935.41242.8C43H68O22936.4202317, 479, 641, 773, 935Rebaudioside F29.451097.4677[M-H] - 1097.46522.3C49H78O271098.4731317, 479, 641, 773, 1097Reb F + 1 Glucose310.921259.5228[M-H] -1259.51813.8C55H88O321260.5259317, 479, 641, 773, 1259Reb F + 2 Glucose411.911421.5750[M-H] - 1421.57092.9C61H98O371422.5787317, 479, 641, 773, 1421Reb F + 3 Glucose512.671583.6261[M-H] - 1583.62371.5C67H108O421584.6315317, 479, 641, 773, 809, 1583Reb F + 4 Glucose613.441745.6788[M-H] - 1745.67651.3C73H118O471746.6844317, 479, 641, 773, 971, 1745Reb F + 5 Glucose713.99953.3607[M-2H] 2- 953.3608-0.1C79H128O521908.7372317, 479, 641, 773, 953, 1133Reb F + 6 Glucose814.691034.3903[M-2H] 2- 1034.38723.0C85H138O572070.7900317, 479, 641, 773, 1034, 1295Reb F + 7 Glucose915.101115.4133[M-2H] 2- 1115.4136-0.3C91H148O622232.8428317, 479, 641, 773, 1115, 1457Reb F + 8 Glucose1015.531196.4402[M-2H] 2- 1196.44000.2C97H158O672394.8957317, 479, 641, 773, 1196, 1619Reb F + 9 Glucose1115.891277.4659[M-2H] 2- 1277.4664-0.4C103H168O722556.9485317, 479, 641, 773, 1277, 1781Reb F + 10 Glucose1216.181358.4943[M-2H] 2-1358.49281.1C109H178O772719.0013317, 479, 641, 773, 1358, 1943Reb F + 11 Glucose

[0149] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification16.01803.3707[M-H] - 803.37010.7C38H60O18804.3780317, 479, 641, 803Stevioside29.01965.4245[M-H] - 965.42301.6C44H70O23966.4308317, 479, 641, 803, 965STV + 1 Glucose310.711127.4762[M-H] - 1127.47580.4C50H80O281128.4836317, 479, 641, 803, 1127STV + 2 Glucose411.821289.5328[M-H] - 1289.52863.2C56H90O331290.5364317, 479, 641, 803, 1289STV + 3 Glucose512.581451.5868[M-H] - 1451.58143.7C62H100O381452.5893317, 479, 641, 803, 1451STV + 4 Glucose613.401613.6406[M-H] - 1613.63433.9C68H110O431614.6421317, 479, 641, 803, 971, 1613STV + 5 Glucose714.041775.6921[M-H] - 1775.68712.8C74H120O481776.6949317, 479, 641, 803, 1133, 1775STV + 6 Glucose814.62968.3677[M-2H] 2-968.36601.7C80H130O531938.7477317, 479, 641, 803, 968, 1295STV + 7 Glucose915.151049.3926[M-2H] 2- 1049.39250.1C86H140O582100.8006317, 479, 641, 803, 1049, 1457STV + 8 Glucose1015.611130.4205[M-2H] 2- 1130.41891.4C92H150O632262.8534317, 479, 641, 803, 1130, 1457, 1619STV + 9 Glucose1115.931211.4451[M-2H] 2- 1211.4453-0.2C98H160O682424.9062317, 479, 641, 803, 1211, 1619, 1781STV + 10 Glucose1216.251292.4722[M-2H] 2- 1292.47170.4C104H170O732586.9590317, 479, 641, 803, 1292, 1781, 1943STV + 11 Glucose

[0150] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification14.50787.3735[M-H] - 787.3752-2.2C38H60O17788.3831317, 479, 625, 787Dulcoside A28.15949.4290[M-H] - 949.42811.0C44H70O22950.4359317, 479, 625, 949Dul A + 1 Glucose39.901111.4838[M-H] - 1111.48092.6C50H80O271112.4887317, 479, 625, 1111Dul A + 2 Glucose411.151273.5363[M-H]- 1273.53372.0C56H90O321274.5415317, 479, 625, 1273Dul A + 3 Glucose512.101435.5874[MH] - 1435.58650.6C62H100O371436.5944317, 479, 625, 809, 1435Dul A + 4 Glucose613.041597.6460[MH] - 1597.63944.2C68H110O421598.6472317, 479, 625, 971, 1597Dul A + 5 Glucose713.641759.6954[MH] - 1759.69221.8C74H120O471760.7000317, 479, 625, 1133, 1759Dul A + 6 Glucose814.22960.3688[M-2H] 2- 960.36860.2C80H130O521922.7528317, 479, 625, 960, 1295Dul A+7 Glucose914.751041.3922[M-2H] 2- 1041.3950-2.7C86H140O572084.8057317, 479, 625, 1041, 1457Dul A+8 Glucose1015.241122.4227[M-2H] 2- 1122.42141.1C92H150O622246.8585317, 479, 625, 1122, 1619Dul A + 9 Glucose1115.641203.4487[M-2H] 2- 1203.44780.7C98H160O672408.9113317, 479, 625, 1203, 1781Dul A + 10 Glucose1215.961284.4775[M-2H] 2- 1284.47422.5C104H170O722570.9641317, 479, 625, 1284, 1943Dul A + 11 Glucose

[0151] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification13.60641.3181[M-H] - 641.31731.2C32H50O13642.3251317, 479, 641Rubusoside27.17803.3665[M-H] - 803.3701-4.5C38H60O18804.3780317, 479, 641, 803Rubu + 1 Glucose39.37965.4227[M-H] - 965.4230-0.3C44H70O23966.4308317, 479, 641, 965Rubu + 2 Glucose410.751127.4775[M-H] - 1127.47581.5C50H80O281128.4836317, 479, 641, 1127Rubu + 3 Glucose511.781289.5319[M-H] - 1289.52862.5C56H90O331290.5364317, 479, 641, 809, 1289Rubu + 4 Glucose612.461451.5875[M-H] - 1451.58144.2C62H100O381452.5893317, 479, 641, 809, 971, 1451Rubu + 5 Glucose713.421613.6345[M-H] - 1613.63430.1C68H110O431614.6421317, 479, 641, 803, 971, 1133, 1613Rubu + 6 Glucose814.041775.6855[M-H] - 1775.6871-0.9C74H120O481776.6949317, 479, 641, 803, 1133, 1295, 1775Rubu + 7 Glucose914.75968.3644[M-2H] 2-968.3660-1.7C80H130O531938.7477317, 479, 641, 803, 968, 1295, 1457Rubu + 8 Glucose1015.131049.3920[M-2H] 2- 1049.3925-0.4C86H140O582100.8006317, 479, 641, 1049, 1457Rubu + 9 Glucose1115.411130.4196[M-2H] 2- 1130.41890.6C92H150O632262.8534317, 479, 641, 803, 1130, 1619Rubu + 10 Glucose1215.911211.4430[M-2H] 2- 1211.4453-1.9C98H160O682424.9062317, 479, 641, 803, 1211, 1781Rubu + 11 Glucose

[0152] Example 7: Nuclear Magnetic Resonance (NMR) Analysis of Glucose-Transferred Steviol Glycosides

[0153] After the reaction solution of the mutant strain prepared in the same manner as in Example 4 was subjected to a glycosylation reaction, the enzyme was inactivated at 100°C, and impurities were removed using a 0.45 μm filter. The reaction solution from which the impurities had been removed was then loaded onto a column filled with an adsorption resin (LXS-869, Sunresin), eluted using 70% ethanol, and the eluent was passed through an anion exchange resin (LXS-865, Sunresin) and concentrated in vacuo. In order to separate each substance in which 1 to 4 glucoses were transferred to rebaudioside A in the concentrated eluent, the eluent was loaded onto a chromatography column. Using a column packed with C18 resin (ODS-AQ-HG, YMC) and an FPLC system (AKTA avant), each Reb A-G1, Reb A-G2, Reb A-G3, and Reb A-G4 were fractionated, and then evaporated in a dry oven at 105°C to prepare each solid sample of rebaudioside A with 1 to 4 glucose units transferred. Approximately 10 mg of each test sample was dissolved in 600 μL of D2O, filtered, and placed in a high-field NMR sample tube. In order to analyze the binding structure of each separated glucose-transferred rebaudioside A, 1 H / 13 It was confirmed by C NMR, Homonuclear correlation spectroscopy (COSY), Total correlation spectroscopy (TOCSY), Heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC), 2D Rotating frame Nuclear Overhauser Effect Spectroscopy (ROESY), and the results are shown in Tables 11 to 14 below.

[0154] As a result of confirming the structure of a substance with 1 to 4 glucoses transferred to Rebaudioside A (RebA), the substance with 1 glucose transferred was identified as RebA-G1, (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester), and based on the structure of Rebaudioside A (Reb A), a stevioside derivative, it was identified as a structure in which α-D-glucopyranose is α-(1,6) bonded to the 6th position of glucopyranose bonded to the 19th carbon position. (Hereinafter, α-(1,6) bonded α-D-glucopyranose is referred to as "sugar E")

[0155] In addition, the substance in which two glucoses were transferred was identified as RebA-G2a, (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester), which has a structure in which α-D-glucopyranose is α-(1,6) bonded to position 6 of sugar E in RebA-G1. (Hereinafter, α-D-glucopyranose bonded α-(1,6) to sugar E is referred to as "sugar F").

[0156] In addition, the substance with three glucose transfers was identified as RebA-G3a, (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester), which is a structure in which α-D-glucopyranose is bonded α-(1,6) to the 6th position of sugar F in the RebA-G2a structure.

[0157] In addition, the substance with four glucose transfers was identified as RebA-G4a, with the structure of 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester.

[0158] Chemical formula 2 below represents RebA-G1 in which one glucose is transferred to the rebaudioside A; Chemical formula 3 represents RebA-G2a in which two glucoses are transferred to the rebaudioside A; Chemical formula 4 represents RebA-G3a in which three glucoses are transferred to the rebaudioside A; and Chemical formula 5 represents the chemical structure of RebA-G4a in which four glucoses are transferred to the rebaudioside A.

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] Table 11 below is an NMR spectral data interpretation table of Rebaudioside A-G1 (RebA-G1), Table 12 is an NMR spectral data interpretation table of RebA-G2a, Table 13 is an NMR spectral data interpretation table of RebA-G3a, and Table 14 is an NMR spectral data interpretation table of RebA-G4a.

[0166] No.δ H mult. (J in Hz) δ C multHMBC CorrelationCOSY corr.Key ROESY corr.10.80 br m1.81 br m40.0CH239.21.39, 1.75, 1.810.80, 1.39, 1.750.95, 1.1221.39 br m1.75 br m18.6CH20.80, 1.04, 1.75, 1.810.80, 1.04, 1.39, 2.061.8131.04 br t (13.0)2.06 br m ovlp37.3CH227.9, 178.91.39, 1.75, 2.061.04, 1.39 w , 2.06 w 1.12, 1.181.18443.8C51.12 br d (11.0)56.6CH15.1, 21.4, 27.9, 39.2, 43.8, 178.91.76, 1.810.80, 0.95, 1.04, 1.18, 1.39 (or 1.38), 1.8161.76 br m1.81 br m21.4CH21.12, 1.381.123.33, 3.4371.38 br m1.51 br m40.7CH253.1, 56.61.51, 1.761.38,1.183.33, 3.43841.9C90.95br d (8.0)53.1CH15.1, 20.1, 36.4, 39.2, 40.7, 41.9, 44.0, 46.9 w1.560.80, 1.12, 1.38, 1.51, 1.56, 2.001039.2C111.56 br m1.77 br m20.1CH20.95, 1.77, 1.901.56, 1.900.95, 1.902.10121.48 br m1.90 br m36.4CH287.4, 153.81.901.48, 1.56, 1.770.95, 2.10, 3.20, 3.33(or 3.34), 3.43(or 3.44), 3.660.83, 1.44, 2.10 w 4.701387.4C141.44 br m 2.10 br m44.0CH236.4, 41.9, 53.1, 87.441.9, 46.9, 87.4 w 2.101.441.90, 3.33, 3.43, 4.700.83, 1.48, 3.20, 4.70152.00 br d (17.5)2.13 br d (17.5)46.9CH244.053.1, 87.4 w , 153.82.13, 4.87, 5.062.00, 4.87, 5.060.95, 3.33(or 3.34), 3.43(or 3.44), 3.66, 4.851.44, 3.33(or 3.34), 3.43(or 3.44), 3.66, 4.8516153.4C174.87 br s5.06 br s104.5CH246.9, 87.446.9, 87.42.00, 2.132.00, 2.132.00, 2.13, 3.47, 3.66, 4.701.48 w , 3.21, 3.33, 3.59, 3.66, 3.81, 4.70, 4.80181.18 s27.9CH318.6 w , 37.3, 43.8, 56.6, 178.91.04, 1.12, 1.81, 2.0619178.9C200.83 s15.1CH339.2, 40.0, 53.1, 56.61.12, 1.75, 1.81, 1.90, 2.10, 3.203.33, 3.43, 3.59, 3.661'5.40 br d (8.0)94.1CH75.3, 76.3, 178.93.421.81 w, 2.06 w , 3.42, 3.49, 3.682'3.42 br m71.9CH76.3, 94.13.30~3.49 / 3.64~3.843'3.49 br m*76.3 a CH69.0, 71.95.404'3.48 br m*69.0CH65.2, 75.35'3.68 e br m*75.3 b CH5.406'3.67 e br m*3.89 br dd (11.0, 4.0)65.2CH297.897.83.893.673.49, 4.851''4.70 ovlp solv.95.9CH75.2, 78.7, 85.0, 87.43.661.44, 1.90, 2.10, 3.33, 3.822''3.66 br m78.7CH85.0, 95.9, 102.14.703''3.82 br m85.0CH68.6, 78.7, 95.9 w , 102.23.433.33, 4.704''3.43 br m68.6CH60.8, 85.03.30~3.49 / 3.64~3.845''3.33 f br m75.2 b CH60.83.43, 3.64, 3.79 w 3.82, 4.706''3.64 g br m*3.79 br m*60.8 c CH23.33, 3.793.641'''4.80 br d (8.0)102.1CH76.4, 78.73.203.32, 3.39, 3.66, 5.062'''3.20 br t (8.0)74.2CH76.1, 102.13.39, 4.803'''3.39 br m76.1 d CH70.2, 74.2, 102.1 w 3.20, 3.214.804'''3.21 br t (8.5)70.2CH61.5, 76.43.32, 3.393.59, 3.815'''3.32 f br m76.4 aCH3.21, 3.594.806'''3.59 br m3.81 br m*61.4CH276.43.32, 3.813.591''''4.72 br d (8.5)102.2CH73.4, 75.8, 85.03.303.33, 3.44, 3.822''''3.30 br t (8.5)73.4CH75.8, 102.23.44, 4.723''''3.44 h br m75.8 d CH69.5, 102.2 w 3.30~3.49 / 3.64~3.844''''3.34 f br m69.5CH5''''3.33 h br m75.7 d CH3.30~3.49 / 3.64~3.846''''3.64 br m*3.84 br m60.6 c CH269.53.843.43, 3.641'''''4.85 br d (4.0)97.8CH65.2, 71.7, 73.13.463.46, 3.67, 3.892'''''3.46 br m71.5CH97.8,3.65, 4.853'''''3.65 g br m*73.1CH69.3, 71.53.36, 3.464'''''3.36 br m69.3CH60.4, 71.7, 73.13.61, 3.655'''''3.61 br m71.7CH3.36, 3.68, 3.816'''''3.68 e br m3.81 br m*60.4 c CH23.61

[0167] W Weak signals. * The chemical shift of each signal may not be exact value due to signal overlapping a,b,c,d,e,f,g,h The assignment of signals at same alphabet column may be exchangeable.

[0168]

[0169] No.δH mult. (J in Hz)δ C multHMBC CorrelationCOSY corr.Key ROESY corr.10.80 br m1.82 br m39.9CH21.39, 1.73, 1.820.800.96, 1.05, 1.131.3921.39 br m1.73 br m18.6CH20.80, 1.05, 1.730.80, 1.391.052.05 w 31.05 br m2.05 br m37.2CH2179.11.39, 1.73, 2.051.050.80, 1.18, 1.39 w 1.18, 1.39443.8C51.13 br d (11.5)56.5CH15.1, 21.4, 39.1, 43.8, 179.11.76, 1.800.80, 0.96, 1.05, 1.18, 1.39, 1.8061.76 br m1.80 br m21.4CH21.13, 1.39, 1.801.13, 1.39, 1.761.491.1871.39 br m1.49 br m40.7CH256.51.491.390.96, 1.13, 1.800.96, 1.76841.9C90.96 br d (7.5)53.0CH15.1, 20.0, 36.4, 39.1, 39.9, 41.9, 44.1, 46.81.550.80, 1.13, 1.39, 1.48, 1.55, 2.011039.1C111.55 br m1.75 br m20.0CH241.9, 87.50.96, 1.75, 1.891.55, 1.890.96, 1.892.09121.48 br m1.89 br m36.4CH287.51.55, 1.891.48, 1.750.96, 2.090.83, 2.09, 4.701387.5C141.42 br m 2.09 br m44.1CH236.4, 41.9, 53.0, 87.546.8, 87.5 w2.091.421.89, 4.700.83, 1.48, 1.75, 1.89, 3.20, 4.70152.01 br d (17.5)2.12 br d (17.5)46.8CH2153.52.12, 4.87, 5.042.01, 4.87, 5.040.96, 3.35, 3.44, 3.64, 4.871.42, 1.48, 3.35, 3.44, 3.64, 4.8716153.5C174.87 br s 5.05 br s104.3CH246.8, 87.546.8, 87.52.01, 2.122.01, 2.122.01, 2.101.48 w , 3.33(or 3.32), 3.64, 3.80, 4.70, 4.80181.18 s27.9CH337.2, 43.8, 56.5, 179.11.05, 1.13, 1.80, 2.0519179.1C200.83 s15.1CH339.1, 39.9, 53.0, 56.51.75, 1.82, 1.89, 2.09, 3.20, 3.33, 3.43, 3.611'5.41 br d (8.5)94.1CH75.3, 76.3, 179.13.421.80 w , 2.05 w , 3.49, 3.702'3.42 br m71.9CH76.3, 94.15.413'3.49 br m*76.3 a CH69.0, 75.35.414'3.48 br m*69.0CH76.35'3.70 br m75.3 bCH5.416'3.70 br m3.89 br m65.3CH269.0, 97.73.883.683.48, 4.861''4.70 ovlp D2O signal95.9CH75.2, 78.7, 87.53.661.42, 1.89, 2.09, 3.33, 3.822''3.66 br m78.7CH84.9, 95.9, 102.13.82, 4.704.803''3.82 br m84.9CH68.6, 78.7, 102.23.43, 3.663.33, 4.704''3.43 br m68.6CH60.8 75.2, 78.7, 84.93.33, 3.825''3.33 br m75.2 b CH3.43, 3.643.82, 4.706''3.64 br m*3.80 br m*60.8 c CH23.33, 3.803.641'''4.80 br d (8.0)102.1CH75.8, 76.4, 78.73.203.33, 3.38, 3.662'''3.20 t (8.0)74.2CH75.8, 102.13.38, 4.803'''3.38 br m75.8CH70.2, 74.23.20, 3.224.804'''3.22 t (8.5)70.2CH61.3, 75.8, 76.43.33, 3.383.61, 3.815'''3.33 br m76.4 a CH61.3, 70.23.22, 3.614.806'''3.61 br m*3.81 br m*61.3CH23.813.33, 3.611''''4.71 ovlp D2O signal102.2CH73.4, 75.8, 84.93.303.33, 3.44, 3.822''''3.30 br t (8.5)73.4CH75.8, 102.23.44, 4.713''''3.44 br m75.8 d CH102.23.304''''3.35 br m*69.5 e CH5''''3.33 br m*76.1 d CH60.63.656''''3.65 br m*3.84 br m*60.6 cCH23.33, 3.823.651'''''4.86 br s97.7CH65.3, 70.2, 71.4(or 71.5), 73.4(or 73.1)3.483.48, 3.70, 3.892'''''3.48 br m71.4 f CH73.44.863'''''3.64 br m*73.4 g CH69.3(or 69.4), 71.5(or 71.4)4'''''3.43 br m* 69.4 e CH65.33.805'''''3.80 br m*70.2CH3.436'''''3.63 br m*3.89 br m65.3CH297.797.71''''''4.87 br s97.7CH65.3, 71.8, 71.4(or 71.5), 73.4(or 73.1)3.473.47, 3.63, 3.892''''''3.47 br m71.5 f CH4.873''''''3.64 br m*73.1 g CH69.3(or 69.4), 71.5(or 71.4)4''''''3.36 br m*69.3 e CH60.43.63(or 3.64)5''''''3.63 br m*71.8CH6''''''3.68 br m3.76 br m60.4CH2

[0170]

[0171] No.δ H mult. (J in Hz)δ C multHMBC CorrelationCOSY corr.Key ROESY corr.10.81 br m1.81 br m39.9CH239.1, 52.91.39, 1.72, 1.810.810.96, 1.14, 1.810.81, 0.82, 1.3921.39 br m1.72 br m18.5CH20.81, 1.06, 1.720.81, 1.06, 1.39, 1.81, 2.040.96, 1.06, 1.812.04 w31.06 br m2.04 br m37.1CH2179.11.39, 1.72, 2.041.06, 1.39, 1.720.81, 1.14, 1.39 w1.18, 1.39, 1.72443.8C51.14 br d (11.5)56.4CH15.1, 21.4, 27.9, 39.1, 43.8, 179.11.75, 1.800.81, 0.96, 1.06, 1.39, 1.8061.75 br m1.80 br m21.4CH21.14, 1.391.14, 1.391.481.1471.39 br m1.48 br m40.6CH21.48, 1.75, 1.801.390.96, 1.140.96, 1.75841.9C90.96 br d (8.0)52.9CH15.1, 20.0, 36.4, 39.1, 39.9, 40.6, 41.9, 44.1, 46.81.560.81, 1.14, 1.39, 1.48, 1.56, 2.021039.1C111.56 br m1.77 br m20.0CH239.139.1, 41.9, 87.60.96, 1.77, 1.901.56, 1.900.96, 1.902.09121.48 br m1.90 br m36.4CH287.6, 153.61.77, 1.901.48, 1.56, 1.770.96, 2.090.82, 1.42, 4.711387.6C141.42 br m 2.09 br m44.1CH236.4, 41.9, 52.9, 87.646.8, 87.6, 153.62.091.421.90, 4.710.82, 1.48, 1.77, 1.90, 4.71152.02 br d (17.0)2.12 br d (17.0)46.8CH244.1, 52.9153.62.12, 4.87, 5.042.02, 4.87, 5.040.96, 1.42, 1.56, 3.33-3.35, 3.64(or 3.65), 4.871.42, 1.48, 3.33- 3.35, 3.64(or 3.65), 4.8716153.6C174.87 br s 5.04 br s104.3CH246.8, 87.646.8, 87.62.02, 2.122.02, 2.122.02, 2.12, 3.821.48 w, 3.33-3.35, 3.64, 3.79, 4.71, 4.80181.18 s27.9CH337.1, 43.8, 56.4, 179.11.06, 1.80, 2.0419179.1C200.82 s15.1CH339.1, 39.9, 52.9, 56.41.14, 1.72, 1.75, 1.81, 1.90, 2.09, 3.20, 3.33, 3.591'5.41 d (8.0)94.1CH75.3, 76.2, 179.13.423.49, 3.702'3.42 br m71.9CH76.2, 94.15.413'3.49 br m*76.2 a CH69.05.414'3.49 br m*69.0CH76.25'3.70 br m*75.3 b CH3.495.416'3.70 br m3.91 br m65.4 c CH275.297.73.903.714.874.871''4.71 ovlp D2O signal95.9CH75.8, 78.7 w , 84.9, 87.63.661.42, 1.90, 2.09, 3.33, 3.822''3.66 br m78.7CH84.9, 95.9, 102.14.713''3.82 br m84.9CH68.6, 78.7, 102.23.433.33, 4.714''3.43 br m*68.6CH60.8, 75.2, 78.7, 84.93.33-3.35, 3.825''3.33-3.35 br m*75.2 b CH3.43, 3.644.716''3.64 br m , *3.79 br m*60.8 d CH23.33-3.35, 3.793.641'''4.80 d (8.0)102.1CH75.8, 76.4, 78.73.203.33, 3.38, 3.66, 5.042'''3.20 t (8.0)74.1CH75.8, 102.13.38, 4.803'''3.38 br m75.8 a CH70.2, 74.13.20, 3.224.804'''3.22 t (8.5)70.2 eCH61.3, 76.43.33, 3.383.59, 3.815'''3.33 br m76.4CH3.22, 3.594.806'''3.59 br m3.81 br m61.3CH23.33, 3.813.593.221''''4.72 ovlp D2O signal102.2CH73.4, 75.8, 84.93.303.34, 3.44, 3.822''''3.30 br t (8.5)73.4CH75.8, 102.23.44, 4.723''''3.44 br m*75.8 a CH102.23.304.724''''3.33-3.35 br m*69.5 d CH5''''3.34 br m*76.1 a CH60.63.656''''3.65 br m*3.82 br m*60.6 d CH23.34, 3.823.651'''''4.87 br m*97.7 g CH65.4, 70.1 or 70.2, 71.4 or 71.5, 73.1 or 73.43.483.48, 3.70, 3.912'''''3.48 br m*71.4 f CH97.7(or 97.8)4.873'''''3.65 br m*73.4CH4'''''3.45 br m* 69.5 d CH5'''''3.80 br m*70.1 e CH6'''''3.63 br m*3.90 br m65.4 c CH297.7(or 97.8)70.1(or 70.2), 97.81''''''4.87 br m*97.7 g CH65.4, 70.1 or 70.2, 71.4 or 71.5, 73.1 or 73.43.483.48, 3.63, 3.902''''''3.48 br m*71.4 f CH97.7(or 97.8)4.873''''''3.66 br m* 73.4CH4''''''3.44 br m*69.4 d CH3.805''''''3.80 br m70.2e CH23.436''''''3.63 br m*3.90 br m65.3 c CH297.8(or 97.7)70.1(or 70.2), 97.81'''''''4.87 br m*97.8 g 65.3, 70.1 or 70.2, 71.4 or 71.5, 73.1 or 73.43.493.49, 3.63, 3.902'''''''3.49 br m*71.5 f CH97.8(or 97.7)4.873'''''''3.65 br m*73.1CH4'''''''3.36 br m*69.4 d CH3.64-3.655'''''''3.64 br m*71.8CH6'''''''3.70 br m3.75 br m60.4CH2

[0172]

[0173] o.δ H mult. (J in Hz)δ C multHMBC CorrelationCOSY corr.Key ROESY corr.10.82 br m1.81 br m39.7CH21.38, 1.810.820.97, 1.16, 1.811.17, 1.38,21.38 br m1.70 br m18.5CH20.82, 1.07 w, 1.700.82, 1.38, 2.031.81, 2.030.82, 2.0331.07 br td (13.5, 4.0)2.03 br d (13.5)37.1CH2179.21.38, 1.70, 2.031.03, 1.38, 1.701.17, 1.381.16, 1.38, 1.70443.8C51.16 br d (12.5)56.3CH15.1, 21.3, 37.1, 39.1, 40.6, 43.8, 179.21.74, 1.810.82, 0.97, 1.07, 1.4161.74 br m1.81 br m21.3CH21.16, 1.410.82, 1.47, 3.45, 3.64, 3.803.45, 3.64, 3.8071.41 br m1.47 br m40.6CH21.47, 1.74, 1.811.41, 1.740.97, 1.16841.8C90.97 br d (8.0)52.7CH15.1, 19.9, 39.1, 41.8, 44.11.560.82, 1.16, 1.38 w , 1.48 w , 1.56, 2.031039.1C111.56 br m1.76 br m19.9CH239.141.8, 87.60.97, 1.76, 1.891.56, 1.890.97, 1.891.48121.48 br m1.89 br m36.4CH287.61.56, 1.891.48, 1.761.76, 2.100.82, 2.10, 4.721387.6C141.42 br m 2.10 br d (13.0)44.1CH287.646.72.101.424.720.82, 0.97, 1.76, 1.89, 4.72 w152.03 br d (15.5)2.11 br d (15.5)46.7CH2153.7153.72.10, 4.87, 5.032.02, 4.87, 5.030.97, 1.56, 3.32- 3.35, 3.64(or 3.65), 4.871.42, 1.48, 3.32- 3.35, 3.64(or 3.65), 4.8716153.7C174.87 br s 5.03 br s104.2CH246.7, 87.646.7, 87.62.03, 2.112.03, 2.112.03, 2.113.33, 3.59, 3.81, 4.71, 4.80181.17 s27.8CH337.1, 43.8, 56.3, 179.21.07, 1.81, 2.03 w 19179.2C200.82 s15.1CH339.1, 39.7, 52.7, 56.31.16, 1.70, 1.76 1.81, 1.89, 2.10, 3.22 w 1'5.41 d (8.0)94.1CH75.3, 76.3, 179.23.423.49, 3.702'3.42 br m71.9CH76.2, 94.15.413'3.49 br m*76.2 a CH69.05.414'3.49 br m*69.0CH76.23.705'3.70 br m*75.3 b CH69.03.495.416'3.70 br m3.90 br m65.4 c CH275.397.73.903.704.884.881''4.72 ovlp D2O signal95.9CH78.7, 84.9, 87.73.661.42, 1.89, 2.10, 3.33, 3.822''3.66 br m78.7CH84.9, 95.9, 102.03.82, 4.723''3.82 br m84.9CH68.6, 78.7, 102.23.43, 3.664.724''3.43 br m*68.6CH60.8, 75.2, 78.7, 84.93.32-3.35, 3.825''3.32-3.35 br m*75.2 bCH3.43, 3.644.726''3.64 br m*3.79 br m*60.8 d CH23.32-3.35, 3.793.641'''4.80 d (8.0)102.0CH75.8, 76.3 78.73.203.33, 3.38, 3.662'''3.20 t (8.0)74.1CH75.8, 102.13.38, 4.803'''3.38 br m75.8 a CH70.2, 74.13.20, 3.224.804'''3.22 t (8.5)70.2 e CH61.3, 75.8, 76.43.33, 3.383.59, 3.815'''3.33 br m76.4CH61.33.22, 3.594.806'''3.59 br m3.81 br m61.3CH276.43.33, 3.813.33, 3.591''''4.71 ovlp D2O signal102.2CH73.4, 75.8, 84.93.303.33, 3.44, 3.822''''3.30 br t (8.5)73.4CH75.8, 102.23.44, 4.713''''3.44 br m*75.8 a CH3.304.714''''3.35 br m*69.4 d CH75.8, 76.15''''3.32-3.33 br m*76.1 a CH60.63.656''''3.65 br m*3.82 br m*60.6 d CH269.43.32-3.33, 3.823.651'''''4.86-4.90 br m*97.7 g CH65.4, 70.0 or 70.2, 71.4 or 71.5, 73.1 or 73.43.44-3.503.44-3.50, 3.902'''''3.44-3.50 br m*71.4 f CH4.86-4.903'''''3.61-3.67 br m*73.4CH71.44'''''3.45 br m* 69.5 d CH65.45'''''3.80 br m70.0 eCH6'''''3.62-3.65 br m*3.88-3.92 br m*65.4 c CH297.870.0, 97.81''''''4.86-4.90 br m*97.8 g CH65.4, 70.0 or 70.2, 71.4 or 71.5, 73.1 or 73.43.44-3.503.44-3.50, 3.62- 3.65, 3.88-3.922''''''3.44-3.50 br m*71.4 f CH4.86-4.903''''''3.66 br m* 73.4CH71.44''''''3.44 br m*69.4 d CH65.43.805''''''3.80 br m*70.2 e CH23.446''''''3.62-3.65 br m*3.88-3.92 br m*65.4 c CH297.870.2, 97.81'''''''4.86-4.90 br m*97.8 g CH65.4, 70.0 or 70.2, 71.4 or 71.5, 73.1 or 73.43.44-3.503.44-3.50, 3.62- 3.65, 3.88-3.922'''''''3.44-3.50 br m*71.4 f CH4.86-4.903'''''''3.66 br m* 73.4CH69.4(or 69.5), 71.4(or 71.5)4'''''''3.44 br m*69.4 d CH65.33.805'''''''3.80 br m70.2 e CH23.446'''''''3.62-3.65 br m*3.88-3.92 br m*65.3 c CH297.670.2, 97.61''''''''4.86-4.90 br m*97.6 g65.3, 70.0 or 70.2, 71.4 or 71.5, 73.1 or 73.43.44-3.503.44-3.50, 3.62- 3.65, 3.88-3.922'''''''3.44-3.50 m*751. f CH4.86-4.903'''''''3.65 br m*73.1CH69.5(or 69.4), 71.5(or 71.4)4''''''''3.36 br m*69.5 d CH71.83.64-3.655''''''''3.64 br m*71.8CH6'''''''3.69 br m3.76 br m60.4CH2

[0174]

[0175] [서열번호 1]:

[0176] taaatcgaga gtttgatcct ggctcaggac gaacgctggc ggcgtgccta atacatgcaa 60

[0177] gtcgaacgca aaactttcat cgaatgcttg cattcaccga aagttttgag tggcgaacgg 120

[0178] gtgagtaaca cgtgggtaac ctgcccagaa gagggggata acacttggaa acaggtgcta 180

[0179] ataccgcata acaataaaaa ccgcatggtt tttatttaaa agatggtttt gctatcactt 240

[0180] ctggatggac ccgcggcgta ttagctagtt ggtaaggtaa aggcttacca aggcaatgat 300

[0181] acgtagccga actgagaggt tgatcggcca cattgggact gagacacggc ccaaactcct 360

[0182] acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga gcaacgccgc 420

[0183] gtgagtgaag aaggttttcg gatcgtaaaa ctctgttgtt agaagaac gtgtgtgaga 480

[0184] gtaactgctc atgcagtgac ggtatctaac cagaaagcca cggctacta cgtgccagca 540

[0185] gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa agggaacgca 600

[0186] ggcggttttt taagtctgat gtgaaagcct tcggcttaac cgaagtcatg cattggaaac 660

[0187] 720

[0188] atatatgaa gaacaccagt ggcgaaagcg gctctctggt ctgtaactga cgctgaggtt 780

[0189] cgaaagtgtg ggtagcaaac aggattagat accctggtag tccacaccgt aaacgaatgaa 840

[0190] tgctaagtgt tggagggttt ccgcccttcg gtgctccagc taacgcatta agcattccgc 900

[0191] ctggggagta cgaccgcaag gttgaaactc aaaggaattg acggggccc gcacaagcgg 960

[0192] tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcttct 1020

[0193] gacaacctaa gagattaggt gttcccttcg gggacagaat gacaggtggt gcatggttgt 1080

[0194] cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttattatt 1140

[0195] agttgccagc attaagttgg gcactctagt gagactgccg gtgacaaacc ggaggaaggt 1200

[0196] ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1260

[0197] cggtacaacg agtcgcgaaa ccgcgaggtt tagctaatct cttaaagccg ttctcagttc 1320

[0198] ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg cggatcagca 1380

[0199] tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgagagtttg 1440

[0200] taacacccaa agccggtgag gtaaccttta tgggaccagc cgtctaaggt gggacagatg 1500

[0201] attggggtga agtcgtaaca aggtagccgt aggagaacct gcggctggat cacctccttt 1560

[0202]

[0203] [서열번호 2] :

[0204] atgcgctcga caaccacata tgcagttgtt gatattgaga cgactggcac aagtagtgat 60

[0205] ggcagcaata ggatgctgca gttcagctgt gtttttataa aaaataaaga aatagttaat 120

[0206] acatttaata caatgattaa tcctgggatg ccaattccca ttgaagttca aaaattaaca 180

[0207] ggtattagtg acaaaaaatgt caggaaagca cctttttttg aagatatggc aggtacaatt 240

[0208] tattctttac tgcaaggaac agtgtttatt gcgcataata tcaattttga ttatcgtttt 300

[0209] ttaaacgaag agtttttgcg ttgcggctat ccagaactaa atattcatgg aattgatact 360

[0210] gtacagttga gtcaaattgt tttacctacc ttaccaagct atcgtctaac atatcttggt 420

[0211] gagtatttcg atatcagaca tgaacaccca caccacgctg atagtaatgc ttttgttacg 480

[0212] gcaaaacttt ttttgatgct gttaaaggca attgataatc taccagttca ggtcttgaga 540

[0213] ataattaata ggtttagtga aagtctcctt tttcaaacag gatcttgttt tgctgctgct 600

[0214] cttaaaaaa agcaagcgca gacaaaacag ctgccgaact atcttgaagt tgttggtgat

[0215] ttagtattaa gacggaac actgacaatt gagagcatga gagagggtgg ttatccgcaa

[0216] actcgtgaga aaaaggaaa cttgtttggt aagttcttgg aatggcgacc aacacaatca

[0217] gaaatgatgg acgaggtata tcgtttatta gtacagcgaa aagaaaagct gcttatgatt

[0218] gaggcaccga ctggattagg caagacatta ggatatctga tacctgctct atatgctgca

[0219] gtcaaaggac acccaagtgt agtatcgaca gcgactacaa ctttacaaat gcagttgttg

[0220] gacagacta ttccactgct tagacaata atgccattta attttacggt agctgttttg

[0221] aaggggagcc aatattacat agatttacag aaatttgctt tgtctctggg caagccgcag

[0222] aataaccat cgaggttatt acagctgcgc atagttgttt ggctaacaat gactaagacc

[0223] ggcgatttga gtgaactcca tctgactaaa atgcaagacc cacttttcga tgacataaca

[0224] cataaaggac cgttgagcat tgatagtggc agtgtttat acacacatga ttttgtgtta 1260

[0225] aggcaacagt tgaaacaagc ctcggccgat ttgataatta caaatcactc ctatctgtta 1320

[0226] aatcatgctg aggggctcgg aagatttaag aagaaatcat tgattattga cgaagcacag 1380

[0227] cactttggaa gtatcgcgtt aaagagtaac cgcgcagtca ttgattttga tttgatcaaa 1440

[0228] atcatttcag acacactttt agttaaaatc ggctcacaaa ggtcattttc attcaaggag 1500

[0229] ttagagcaac agtattttct gacaccggcc gagtcgaaaa aaattgcagc acagatcaga 1560

[0230] gtaatcgata agagagtacc agcgttaaga gagttattac gcagtagatt cctccagaaa 1620

[0231] gagaaaaaag aaaatgggga ggatgctttt aatgaagttg ctgtaaagac aagtaagttc 1680

[0232] cagggatttg tgaaagaaaa tcttgcagat taccaaaaag ttgccaaggc taaagcaaaa 1740

[0233] tttcaggtac agctattgca gttaaagaca aaatttattg aattcaaaaa tcaagaaagg 1800

[0234] ttggatcgaa atgcgcaac atttgtacta gatttctctg atggtggttt tgaactttta 1860

[0235] aaagcattgg agactggca tcgatttgaa cttgatgaac tgatcagat agctgaggaa 1920

[0236] acagtaatta gtctgcaat tccagtgaaa caataatg gtcatttacg gcttagtttt 1980

[0237] ggaattttta aaaaaayata ctatctctcg ccttttgttt attcgaagtt tgagcataca 2040

[0238] ctttttgttg gtgctgcgtt agttttacct gaagggtcag actatatgaa aaaccagctt 2100

[0239] gatttagcat ctgagacacc tgtattaaga ctgaaggtg gtttgacta tcataacaa 2160

[0240] gcattgggat tgttagtcgc tgatgctcct gatatcgtta cagacactga aaggtacatt 2220

[0241] gcgtatttag gtaagttat agaggacatt ttagcaata ataatagca aactatgatt 2280

[0242] ttgtttaatt cacttgaaat gatttctaaa gtctatgatt atttacgtca atcagaagtt 2340

[0243] tttgacaaaa gagtgattat tgctcagga attackaggca gcaatgaaaa gatatcaag 2400

[0244] atgtttgaat taggtgacaa tgcggtcttg cttggttcgg gtactttctg ggagggaatc 2460

[0245] gatctgccta aagatcgatt ggaacttctg gttattacac gattgccatt tcaaccgccg 2520

[0246] aatacattgg tcaatcgtgc aaagtatcgg ctggcacaaa gtcaaggtca agattcattt 2580

[0247] aatacgattg cgttgccgga agcgatgttt cgtttaaagc aaggccttgg aagattaatc 2640

[0248] agaacgaagg aagacagagg agtcgtaatt gttttagata gccgtgtggt ttcaagaaac 2700

[0249] tatggtgcta aattgcgaga ggtctttcca catgagatgc cggtaagaat tatcgagtca 2760

[0250] actgaaattc agaattattt gtttgatttt tgggaaaatt ag 2802

[0251]

[0252] [서열번호 3] :

[0253] atgggtggtg cagcacttca agtgattagc agttatagtt tgctgcaaag ttcaattcga 60

[0254] ttaaaagatg tcattaaaag tgctaaagca aagggatata cagcattagc tttaacagat 120

[0255] atcaatgtta tgtacggagt cctttcattt tatgatgcat gtcgagttga aggcattaaa 180

[0256] ccacttatag ggatgacact agagacatcc accgacaaaa aagaaacact gattttaatt 240

[0257] gctaaaaacg agaacggtta tcacaacttg attcagattt cgactcaaaa acaattgtta 300

[0258] cttgcaaaga atgatgcaga attcataatc agggaaaatg ccgctctttt tgaagatata 360

[0259] attacgataa ttccacctga ggatagtttt tttgtcaaca agctattgaa tggagagagt 420

[0260] gaacttgcaa gtaaatatct gaaacaaagt agaaatttat tgcaagaaaa tttgtatatt 480

[0261] gggataagta cagcaacatc agaggcactg tatgataaag tattgcaata tgcaaataaa 540

[0262] atgaatatta agaccgttgc tgttgaatct atcaagtatt tagaaacgaa tgatttgttt 600

[0263] gtttgtaagg tcatgaatgc aattaagcag aataaacagc tagacagtaa agatttgcag 660

[0264] tatgcaaata atctgaatgg ttctgcatgg ctaaaaccgc taaatgacat aaagcaaaac 720

[0265] tatttagaaa aaaatcgttt tgaagcattt gaaaatatga tttctttaac taaacgagtt 780

[0266] gatttcgctt tttcgccaaa aagagtaata cttccaaaat ttcaaacacc gcaagggcatt 840

[0267] 900

[0268] 960

[0269] aagaaaatgg gatttgcaga ctatttctta attgtttggg atgtcacaaa ttatgctcat 1020

[0270] gaagatggaa ttttggttgg tcctggccgt ggctctgcag cgggttcgct ggtttcgtat 1080

[0271] ctcctaggaa ttacagatgt tgacccaatc aaatacaatt tgctttttga acgttttttg 1140

[0272] aatgaagaac gtgcacaaat gcccgatatt gacttggata ttccagataa tcgacgacag 1200

[0273] gaaataattg aatacgttaa tagaaaatat ggtgaaaacc acgtggcaca gataatcact 1260

[0274] tttgggacat ttggtgcaaa acaggctttg cgcgatgttg caagagtaat gggtttttcg 1320

[0275] caagtagaa gtaataattg gagtcgtgta attcccagtc aattgggaat tacactggca 1380

[0276] gatgctgaga agaaatctat tcagttgcgg aatttaatca acgaaaatga gcgcaatcaa

[0277] cttttattta aaactgcgct gctactgga ggcctaccta gacattattc agttcatgct

[0278] gcaggagtga ttctaagtga tgcagatctg agaaaagtag tgccattgca aatgggtaac

[0279] gatgatgttt tgctgacgca atataccaaa gatgatgttg aacgtgtagg attgctgaaa

[0280] attgattttt tagtttaag aaacctaacg attttaaata gtacattgtc tgggataag

[0281] aaaaatttg gccaaaccgt tgatataaat aaaatttcat tgaatgatga agagacgctt

[0282] agtctttttc aaagggccga tactagcggt gtatttcagt ttgaatcatc cggtattcgc

[0283] aatgttttgc gtaatcttta cccaacttct tttgaggata ttgctgctgt aaatgcattg

[0284] tttagacctg gtcctatgga gaatcaca cactttatag ctcgtaaaca tggtaaggag

[0285] aagattgttt atccagataa ttcgctaatt ccgatcctga agaatacgta tgggattttg

[0286] gtttatcagg agcaagttat gcaagttgct tctataatgg gtggatttac tttagggcaa 2040

[0287] gccgatatat tgcgaagagc aatgagtaaa aagaagcta gtgtattga agaattgaag 2100

[0288] tccaagtttg tcgatggcgc tgtaagttg ggatattctc aaaaaacgc agaacgagtc 2160

[0289] tacgattata tgaacgttt tgctattat ggatttaacc gttcgcatgc cattgcatat 2220

[0290] tctaaaatag cattcagct tgcctattta aaagttcact atgctgcgcc ttttttgca 2280

[0291] gcaattttga attctgttat cggtgacaga aagacaa gagatttt agttgagca 2340

[0292] aagcagcatc attgcagat tgagactcca atataaca ggagcaatta ttatactttc 2400

[0293] tctgggaaaa agaacttgat tattggttg ggcaatca agaccttgcg cagagatttt 2460

[0294] atcaaagaaa tcattgaaga acgtaagaat acaggtcgtt atagttcgtt tgatgacttt 2520

[0295] atagagaa ttaatcacaa gttttaaaa gaggagccac ttaggcttt gatatattct 2580

[0296] ggcacttttg actcgttttc tgaaaatcgt gcaactttat tgggcaatct tagtaaaaaag 2640

[0297] atgagcaatg ttgagttgag tggagagagc agtgagttgt tatcattact agcacccaag 2700

[0298] caggaaaaat atacagaact tcccttggaa gagatttag ccggagagca gaagtatctt 2760

[0299] ggaattttcc tttcggctca tccagtagag cagtttgcgg aggttgcaca gctgcataac 2820

[0300] gcaaagatga tagcaaatgt tcacgaaaat gagaagcagc aaattttgtg tttggttaag 2880

[0301] aagataaaag taattagaac aaaaactggt caacaaatgg cttttttaac ggtagaggat 2940

[0302] caaacaggcg aaatcgaatt gacgcttttt ccaggtattt ttgaaaaggt tgatgatgat 3000

[0303] ttgaaaacaa atcaagttta tcttgtttct ggaaagagtg aaaaaggcaa tcaaaagata 3060

[0304] caaattatcg tttcaacgat ggtttcagct gaaagtttga aaacagaaat gaaaggaaga 3120

[0305] ttatttttaa gactaactga aaatgattca agcagtataa acaaacaact tttgaaaaca 3180

[0306] ttacagttac atgctggaaa agttccagtt gtcttgtatg aggagaaaaa agccgtgaaa 3240

[0307] tgggttttag atgaaaagta ctgggtaaac aggtcgccag aattagaaga acagttaata 3300

[0308] aagttgattg gtaaaaacaa cgttgtcttt caaaatgaaa acaaatga 3348

[0309]

[0310] [서열번호 4] :

[0311] ttgaaaaata aaatttaca gctcgaacag aagcacctta atttagtaat agaaaaaatt 60

[0312] agtaaggctg ttaaaaagac ccgaaaaaag ctagcagatt ctaaaaacaa tttagataac 120

[0313] ctcaaaaaga aaaggcca acttaattca ttagaaaaaa tgaaatccaa gccttatttt 180

[0314] gcccgactag actttataga atccgatgaa agtaaaccag aaactattta tataggtaaa 240

[0315] cgaaccttta tcgatgataa tgatttttg gtgtatgatt ggcgtgcccc aatatcctca 300

[0316] atgtactata ctggtgactt aggcaaaact tattataaaa gtccgatggg taaccagaaa 360

[0317] gttgatgtta attaaaact tcaatttaat attaaccatc aaacaattat tatatatat 420

[0318] gatgctaacg catccattgg agatcaatta cttctcgata cgttgtccaa aagttcgtct 480

[0319] aataaaatga aaaatattgt ttcgactatt caaagtgaac agaataaggt tattcgcaac 540

[0320] gatcaagaca atgttttagc cgtgcaggga atcgctggat cggggaaaac agctgtgtta 600

[0321] ttacaacggg tcgcgtggtt gctttatcaa taccgttcaa ccgttaattc aaaacaaatt 660

[0322] ttaattttat caccaaatga acttttcagt agttatatta atggagtatt gccagatttg 720

[0323] ggagagccga atgcgttgca attgactttc actaaattat tcaaagaaaa ttcatgggtc 780

[0324] tcaaattatc acattgaaaa tttaacagag caggccacgc atcagactac caatgctttt 840

[0325] ctaaagagta cggcttgttt taatcgttta gtaaagtatt gtcaattatt aaataagggg 900

[0326] gagtgcagtt ttaaaagtat caaagatggt tctaaatcaa ttatgactag taaccaatta 960

[0327] aaacggatct tctattcatt taacgaaact tataagctat ttaatcgctt tacggccaca 1020

[0328] CAagaacaag ttaaaaata tttggaacga tacctgaata aaatttaata tcaaagtgg 1080

[0329] gttgaagaag aagttggtgc tattaatcca caactttgg ggcttgtgga acgccagcag 1140

[0330] aaatttttcct cattactgc tgacaaat tactggcggc agcagatgt tacgaaaat 1200

[0331] tatcatgaac aaatcgcaca aattaattct gggtcttta tagatattgt agccatagct 1260

[0332] gttaatttt tgaaaagtttt attagactta gtgggaatta gtggtaacca agatgcagtt 1320

[0333] aaatgaaatta atgttgctat tcaaaatta aaggaccaga aaatcgatcc caacttaagt 1380

[0334] gccattttac tatttatcca acagcagttt agtcacgaga ttgccgataa taagatcaag 1440

[0335] ttgttctca ttgatgaat catchaacat accccattc agattgaac catcaaact 1500

[0336] atttatccta aagccaaatt tactttttg ggtgatgcca atcaaatat tttgaaac 1560

[0337] 1620

[0338] actttaaatc gttcttatcg ttcaagtgct cccattacag tctttacaag taatttattg 1680

[0339] ccacaaaata agtttaacca caatattcag tctgttaatc gcactggctt gaagccaaaa 1740

[0340] ttaattacgg taaaccagga taaagatatg ataaaagcat tataagact tattggacta 1800

[0341] aatcaagaca tgcaaattgc tattatttgt aagtccttat cagaagcaca atcgttggtt 1860

[0342] aaaccaataa agaatcagtc cattcgagta gaattagttg cttctgagtt tcagaaaagt 1920

[0343] aatgcaaaaa ttattcttat accagcttat ttagcgaaag gattagagtt tgatactgtt 1980

[0344] attgcttgga atattccca agaacatttt cctggtgaac agcagcggtt gttattatat 2040

[0345] acaatctgtt caagagccat gcatcgcctc ttcttgctca caacagccaa tccttcacct 2100

[0346] ttactttcaa ctattccaag caatctaatc gatcaagaat aa 2142

[0347]

[0348] [서열번호 5] :

[0349] MRSTTTYAVV DIETTGTSMD GSNRMLQFSC VFIKNKEIVN TFNTMINPGM PIPIEVQKLT 60

[0350] GISDKNVRKA PFFEDMAGTI YSLLQGTVFI AHNINFDYRF LNEEFLRCGY PELNIHGIDT 120

[0351] VQLSQIVLPT LPSYRLTYLG EYFDIRHEHP HHADSNAFVT AKLFLMLLKA IDNLPVQVLR 180

[0352] IINRFSESLL FQTGSCFAAA LKKKQAQTKQ LPNYLEVVGD LVLRTETLTI ESMREGGYPQ 240

[0353] TREKKENLFG KFLEWRPTQS EMMDEVYRLL VQRKEKLLMI EAPTGLGKTL GYLIPALYAA 300

[0354] VKGHPSVVST ATTTLQMQLL EQTIPLLRQI MPFNFTVAVL KGSHNYIDLQ KFALSLGKPQ 360

[0355] NKPSRLLQLR IVVWLTMTKT GDLSELHLTK MQDPLFDDIT HKGPLSIDSG SVYYTHDFVL 420

[0356] RQQLKQASAD LIITNHSYLL NHAEGLGRFK KKSLIIDEAQ HFGSIALKSN RAVIDFDLIK 480

[0357] IISDTLLVKI GSQRSFSFKE LEQQYFLTPPA ESKKIAAQIR VIDKRVPALR ELLRSRFLQK 540

[0358] EKKENGEDAF NEVAVKTSKF QGFVKENLAD YQKVAKAKAK FQVQLLQLKT KFIEFKNQER 600

[0359] LDRNAQTFVL DFLDGGFELL KALENWHRFE LDELDQIAEE TVISLQIPVK QINGHLRLSF 660

[0360] GIFKTNNYLS PFVYSKFEHT LFVGAALVLP EGSDYMKNQL DLASETPVLR LEGGFDYHKQ 720

[0361] ALGLLVADAP DIVTDTERYI AYLGKVIEDI LSNNNKQTMI LFNSLEMISK VYDYLRQSEV 780

[0362] FDKRVIIAQG ITGSNEKIIK MFELGDNAVL LGSGTFWEGI DLPKDRLELL VITRLPFQPP 840

[0363] NTLVNRAKYR LAQSQGQDSF NTIALPEAMF RLKQGLGRLI RTKEDRGVVI VLDSRVVSRN 900

[0364] YGAKLREVFP HEMPVRIES TEIQNYLFDF WEN

[0365]

[0366] [서열번호 6]:

[0367] MGGAALQVIS SYSLLQSSIR LKDVIKSAKA KGYTALALTD INVMYGVLSF YDACRVEGIK 60

[0368] PLIGMTLETS TDKKETLILI AKNENGYHNL IQISTQKQLL LAKNDAEFII RENAALFEDI 120

[0369] ITIIPPEDSF FVNKLLNGES ELASKYLKQS RNLLQENLYI GISTATSEAL YDKVLQYANK 180

[0370] MNIKTVAVES IKYLETNDLF VCKVMNAIKQ NKQLDSKDLQ YANNLNGSAW LKPLNDIKQN 240

[0371] YLEKNRFEAF ENMISLTKRV DFAFSPKRVI LPKFQTPQGI NANIYLKQLC SRGMQARMRD 300

[0372] QPQSIIEKYL QRMDKELNII KKMGFADYFL IVWDVTNYAH EHGILVGPGR GSAAGSLVSY 360

[0373] LLGITDVDPI KYNLLFERFL NEERAQMPDI DLDIPDNRRQ EIIEYVNRKY GENHVAQIIT 420

[0374] FGTFGAKQAL RDVARVMGFS QVESNNWSRV IPSQLGITLA DAEKKSIQLR NLINENERNQ 480

[0375] LLFKTALLLE GLPRHYSVHA AGVILSDADL RKVVPLQMGN DDVLLTQYTK DDVERVGLLK 540

[0376] IDFLGLRNLT ILNSTLSGIK KNFGQTVDIN KISLNDEETL SLFQRADTSG VFQFESSGIR 600

[0377] NVLRNLYPTS FEDIAAVNAL FRPGPMENIT HFIARKHGKE KIVYPDNSLI PILKNTYGIL 660

[0378] VYQEQVMQVA SIMGGFTLGQ ADILRRAMSK KKASVIEELK SKFVDGAVKL GYSQKNAERV 720

[0379] YDYIERFANY GFNRSHAIAY SKIAFQLAYL KVHYAAPFFA AILNSVIGDR IKTRDFIVEA 780

[0380] KQHQLQIETP NINRSNYYTF SGKKNLIIGL GNIKTLRRDF IKEIIEERKN TGRYSSFDDF 840

[0381] IRRINHKFLK EEPLKALIYS GTFDSFSENR ATLLGNLSKK MSNVELSGES SELLSLLAPK 900

[0382] QEKYTELPLE EILAGEQKYL GIFLSAHPVE QFAEVAQLHN AKMIANVHEN EKQQILCLVK 960

[0383] KIKVIRTKTG QQMAFLTVED QTGEIELTLF PGIFEKVDDD LKTNQVILVS GKSEKRNQKI 1020

[0384] QIIVSTMVSA ESLKTEMKGR LFLRLTENDS SSINKQLLKT LQLHAGKVPV VLYEEKKAVK 1080

[0385] WVLDEKYWVN RSPLEEEQLI KLIGKNNVVF QNENK 1115

[0386]

[0387] [서열번번 7] :

[0388] MKNKILQLEQ KHLNLVIEKI SKAVKKTRKK LADSKNNLDN LKKKQGQLNS LEKMKSKPYF 60

[0389] ARLDFIESDE SKPETIYIGK RTFIDDNDFL VYDWRAPISS MYYTGDLGKT YYKSPMGNQK 120

[0390] VDVKLKLQFN INHQTIINIY DANASIGDQL LLDTLSKSSS NkmKNIVSTI QSEQNKVIRN 180

[0391] DQDNVLAVQG IAGGSKTAVL LQRVAWLLYQ YRSTVNSKQI LILSPNELFS SYINGVLPDL 240

[0392] GEPNALQLTF TKLFKENSWV SNYHIENLTE QATHQTTNAF LKSTACFNRL VKYCQLLNKG 300

[0393] ECSFKSIKDG SKSIMTSNQL KRIFYSFNET YKLFNRFTAT QEQVKNNLER YLNKIKYQKW 360

[0394] VEEEVGAINP QTLGLVERQQ KFSSITAEQN YWRQQIVTKN YHEQIAQINS GSFIDIVAIA 420

[0395] VNFLKSLLDL VGISGNQDAV NEINVAIQKL KDQKIDPNLS AILLFIQQQF SHEIADNKIK 480

[0396] FVLIDEIQDY TPIQIGTIKT IYPKAKFTFL GDANQNIFEN NYNIFEDINH IFKEDEVKMI 540

[0397] TLNRSYRSSA PITVFTSNLL PQNKFNHNIQ SVNRTGLKPK LITVNQDKDM IKALIRLIGL 600

[0398] NQDMQIAIIC KSLSEAQSLV KPIKNQSIRV ELVASEFQKS NAKIILIPAY LAKGLEFDTV 660

[0399] IAWNISQEHF PGEQQRLLLY TICSRAMHRL FLLTTANPSP LLSTIPSNLI DQE 713

[0400]

[0401] [수탁번호]

[0402] 기탁기관명 : 한국미생물보존센터

[0403] 수탁번호 : KCCM13503P

[0404] 수탁일자: 20240805

[0405]

Claims

Lactobacillus mali strain deposited at the Korean Culture Center of Microorganisms (KCCM) under the accession number KCCM13503P. In claim 1, A Lactobacillus mali strain comprising a DNA nucleotide sequence encoding at least one protein selected from the group consisting of (i) a protein comprising an amino acid sequence of SEQ ID NO: 5; (ii) a protein comprising an amino acid sequence of SEQ ID NO: 6; and (iii) a protein comprising an amino acid sequence of SEQ ID NO:

7. In claim 1, The Lactobacillus mali strain comprises at least one DNA nucleotide sequence selected from the group consisting of (i) a DNA nucleotide sequence encoding 16s rRNA of SEQ ID NO: 1; (ii) a DNA nucleotide sequence of SEQ ID NO: 2; (iii) a DNA nucleotide sequence of SEQ ID NO: 3; and (iv) a DNA nucleotide sequence of SEQ ID NO:

4. In claim 1, The above Lactobacillus mali strain is a Lactobacillus mali strain having a steviol glycoside glucose transfer activity that is 1.4 times greater than that of Lactobacillus mali DSM20444. In claim 1, The Lactobacillus mali strain above has the activity of transferring 1 to 11 glucose molecules to steviol glycosides. In claim 5, A Lactobacillus mali strain, wherein the steviol glycoside is at least one selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, stevioside, dulcoside A, steviolbioside, and rubusoside. A method for producing a glucose-transferred steviol glycoside, comprising the step of contacting the Lactobacillus mali strain of any one of claims 1 to 6, a culture solution or a crude enzyme solution of the strain, with a glucose donor and a steviol glycoside. A composition for producing glucose-transferred steviol glycosides, comprising the Lactobacillus mali strain of any one of claims 1 to 6, or a culture solution or crude enzyme solution of the strain. In claim 8, The composition further comprises a glucose donor and a steviol glycoside. A sweetening composition comprising a glucose-transferred steviol glycoside, The above steviol glycoside comprises at least one selected from the group consisting of rebaudioside A, rebaudioside C, rebaudioside F, stevioside, dulcoside A, and rubusoside, A sweetening composition, wherein the glucose-transferred steviol glycoside comprises 5 to 11 transferred glucose molecules. In claim 10, A sweetening composition, wherein the transfer glucose of the above glucose transfer steviol glycoside is added by linking it to the glucose linked to the 19-OH position of the steviol glycoside by an α-(1,6) bond. In claim 10, A sweet composition, wherein the glucose trans-steviol glycoside is produced by a method for producing a glucose trans-steviol glycoside, comprising the step of contacting the Lactobacillus mali strain of any one of claims 1 to 6, a culture solution or a crude enzyme solution of the strain, with a glucose donor and a steviol glycoside.

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