Novel rebaudioside d and rebaudioside m-like steviol glycosides and method for producing same

Abstract

The present invention relates to novel rebaudioside D and rebaudioside M-like steviol glycosides and a method for producing same. The novel rebaudioside D and rebaudioside M-like steviol glycosides obtained by the production method according to the present invention exhibit improved water solubility, and thus can be easily applied to beverages and foods with a high water content, and can significantly improve the industrial utilization of steviol glycosides.

Description

Novel rebaudioside D and rebaudioside M-like steviol glycosides and methods for producing the same

[0001] The present invention relates to novel Rebaudioside D and Rebaudioside M-like steviol glycosides and a method for producing the same.

[0002] Rebaudioside D and Rebaudioside M, natural sweeteners derived from Stevia rebaudiana, are attracting attention as promising sugar substitutes due to their high sweetness and low calorie content. However, these compounds have low water solubility, limiting their application in beverages and high-moisture foods. This physical property hinders their utility in the food industry.

[0003] In addition, existing chemical or enzymatic conversion methods for producing steviol glycosides have problems such as complex processes, low yields, and low substrate specificity of the enzymes used, making it difficult to apply them to industrial production on a commercial scale.

[0004] Therefore, there is a need for the development of a novel steviol glycoside and an efficient method for producing the same that can improve existing problems.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] Korean Patent No. 10-2797457

[0008] An object of the present invention is to provide a method for efficiently producing Rebaudioside D and M analogues using dextransucrase and UDP-glucotransferase.

[0009] Another object of the present invention is to provide a novel steviol glycoside having improved water solubility of Rebaudioside D and Rebaudioside M.

[0010] Specifically, one object of the present invention is to provide a steviol glycoside represented by the following chemical formula 1:

[0011] [Chemical Formula 1]

[0012]

[0013] Here,

[0014] R1 is or And,

[0015] R2 is , or am.

[0016]

[0017] Another object of the present invention is

[0018] (i) a step of reacting stevioside with dextran sucrase; a microorganism producing the same; or a culture, culture supernatant, lysate or extract thereof of the microorganism; and

[0019] (ii) a step of reacting the reaction product of step (i) with UDP-glucosyltransferase; a microorganism producing the same; or a culture, culture supernatant, lysate or extract thereof of the microorganism;

[0020] A method for producing a steviol glycoside, including:

[0021] One aspect of the present invention is a steviol glycoside represented by the following chemical formula 1:

[0022] [Chemical Formula 1]

[0023]

[0024] Here,

[0025] R1 is or And,

[0026] R2 is , or am.

[0027] The steviol glycoside represented by chemical formula 1 according to the present invention is a steviol glycoside similar to Rebaudioside D and Rebaudioside M, and has increased water solubility compared to Rebaudioside D and Rebaudioside M.

[0028] In one embodiment, in the chemical formula 1

[0029] R1 is And,

[0030] R2 is It can be. Specifically, these steviol glycosides have the following structure and are designated herein as Rebaudioside Mα. Rebaudioside Mα has a water solubility that is more than 100 times higher than Rebaudioside M.

[0031] [Rebaudioside Mα]

[0032]

[0033] The chemical formula of Rebaudioside Mα is O-β-D-glucosyl-(1''→3')-O-α-D-glucosyl-(1'''→6')-O-α-D-glucosyl-(1''''''→6''')-stevioside (C 56 H 90 O 33 )am.

[0034] In this study, R2 glucose is expressed as ', R1 glucose as '''', glucose attached to carbon 3 of R2 glucose as '', glucose attached to carbon 6 as ''', glucose attached to carbon 2 of R1 glucose as ''''', and glucose attached to carbon 6 as ''''''.

[0035] In one embodiment, in the chemical formula 1

[0036] R1 is And,

[0037] R2 is It can be. Specifically, these steviol glycosides have the following structure and are referred to herein as Rebaudioside Mα'. Rebaudioside Mα' has a water solubility that is more than 100 times higher than that of Rebaudioside M.

[0038] [Rebaudioside Mα']

[0039]

[0040] Rebaudioside Mα’ The chemical formula isO-β-D-glucosyl-(1''→3')-O-α-D-glucosyl-(1'''→6')-O-β-D-gluc osyl-(1''''''→3'''')-O-α-D-glucosyl-(1'''''''→6''''')-stevioside (C 62 H 100 O 38 )am.

[0041] In one embodiment, in the chemical formula 1

[0042] R1 is And,

[0043] R2 is It can be. Specifically, these steviol glycosides have the following structure and are named Rebaudioside Dα herein. Rebaudioside Dα has a water solubility that is more than 40 times higher than Rebaudioside D.

[0044] [Rebaudioside Dα]

[0045]

[0046] The chemical formula of Rebaudioside Dα is O-β-D-glucosyl-(1''→2')-O-α-D-glucosyl-(1'''''→6''') stevioside (C 50 H 80 O 28 ) am.

[0047] In one embodiment, in the chemical formula 1

[0048] R1 is And,

[0049] R2 is It could be.

[0050] Specifically, these steviol glycosides have the following structure and are referred to herein as Rebaudioside Dα(3). Rebaudioside Dα(3) is used interchangeably herein with Rebaudioside Dα3.

[0051] [Rebaudioside Dα(3)]

[0052]

[0053] The chemical formula of Rebaudioside Dα3 is O-β-D-glucosyl-(1''→3')-O-α-D-glucosyl-(1'''''→6''') stevioside (C 50 H 80 O 28 ) am.

[0054] In this application, Rebaudioside may be abbreviated as Reb or R.

[0055] Another aspect of the present invention is a sweetener composition comprising a steviol glycoside represented by the chemical formula 1.

[0056] The steviol glycosides according to the present invention are steviol glycosides similar to Rebaudioside D and Rebaudioside M, but have increased water solubility compared to Rebaudioside D or Rebaudioside M. Therefore, the sweetener composition according to the present invention can have increased water solubility compared to a sweetener composition comprising Rebaudioside D or Rebaudioside M.

[0057] The above sweetener composition may further include other natural or artificial sweeteners, etc., as needed.

[0058] In one embodiment, the sweetener composition may be a reaction solution containing a steviol glycoside represented by chemical formula 1 produced by an enzymatic reaction, but is not limited thereto.

[0059]

[0060] Another aspect of the present invention is a method for producing steviol glycosides (e.g., Rebaudioside D and M analogues) using dextransucrase and UDP-glucosyltransferase.

[0061] Specifically, the method for producing steviol glycosides of the present invention is

[0062] (i) a step of reacting stevioside with dextran sucrase; a microorganism producing the same; or a culture, culture supernatant, lysate or extract thereof of the microorganism; and

[0063] (ii) a step of reacting the reaction product of step (i) with UDP-glucosyltransferase; a microorganism producing the same; or a culture, culture supernatant, lysate or extract thereof of the microorganism.

[0064] (i) In step (i), dextran sucrase has the activity of linking glucose to the 13-OH site and / or the 19-OH site of steviol glycosides, including stevioside, via an alpha-1,6 glycosidic bond.

[0065] In the present invention, the dextran sucrase is not limited as long as it has a glycosyltransferase activity of an alpha-1,6 glycosidic bond to a steviol glycoside, but may be a dextran sucrase derived from Leuconostoc kimchi, Leuconostoc citreum, or Leuconostoc lactis.

[0066] The dextran sucrase derived from Leuconostoc kimchi may be a dextran sucrase derived from Leuconostoc kimchii IMSNU11154, and may specifically include an amino acid sequence of SEQ ID NO: 2 or 3, but is not limited thereto.

[0067] The dextran sucrase derived from Leuconostoc citreum may be a dextran sucrase derived from Leuconostoc citreumKM20, and may specifically include the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0068] The dextran sucrase derived from Leuconostoc lactis may be a dextran sucrase derived from Leuconostoc lactis EG001, and may specifically include an amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0069] (i) In step (i), sucrose and / or p-Nitrophenyl-β-D-glucopyranoside (PNPG) may be added as a substrate that provides glucose to the enzymatic reaction of dextran sucrase, but is not limited thereto.

[0070] In the present invention, the reaction temperature of dextran sucrase is not limited as long as its activity is exhibited, but may be 10 to 50°C, 15 to 45°C, 20 to 40°C, 25 to 35°C, 27 to 33°C, or 30°C.

[0071] In the present invention, the reaction pH of dextran sucrase is not limited as long as its activity is exhibited, but may be pH 3 to 8, pH 3.5 to 7.5, pH 4 to 7, pH 4.5 to 6.5, pH 5 to 6, pH 5.3 to 5.8, or pH 5.5.

[0072] In the present invention, the reaction time of dextran sucrase is not limited as long as the target steviol glycoside can be produced, but may be 1 to 72 hours, 6 to 66 hours, 12 to 60 hours, 18 to 54 hours, or 24 to 48 hours.

[0073] (ii) The UDP-glucosyltransferase in step (ii) has an activity capable of linking glucose to the 13-OH site and / or the 19-OH site of the steviol glycoside produced in step (i) via a beta-1,2 or beta-1,3 glycosidic bond.

[0074] In the present invention, the UDP-glucosyltransferase is not limited as long as it has a glycosyltransfer activity of a beta-1,2 or beta-1,3 glycosidic bond to a steviol glycoside, but may be UDP-glucosyltransferase 76G1 (UGT76G1) or UDP-glucosyltransferase 91D2 (UGT91D2).

[0075] The above UDP-glucosyltransferase 76G1 (UGT76G1) is a UDP-glucosyltransferase derived from Stevia (Stevia rebaudiana) and has an activity capable of linking glucose to the 13-OH site and / or the 19-OH site of a steviol glycoside via a beta-1,3 bond. As long as it has the same or corresponding activity as UGT76G1, an enzyme comprising an amino acid sequence having a sequence homology or identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with the amino acid sequence of UGT76G1 is also included in the category of UGT76G1.

[0076] The above UDP-glucosyltransferase 91D2 (UGT91D2) is a UDP-glucosyltransferase derived from Stevia (Stevia rebaudiana) and has an activity capable of linking glucose to the 13-OH site and / or the 19-OH site of a steviol glycoside via a beta-1,2 bond. As long as it has the same or corresponding activity as UGT91D2, an enzyme comprising an amino acid sequence having a sequence homology or identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with the amino acid sequence of UGT91D2 is also included in the category of UGT91D2.

[0077] In the present invention, "homology" or "identity" refers to the degree of relationship between two given amino acid sequences or base sequences, and may be expressed as a percentage. In the present invention, the terms "homology" and "identity" may be used interchangeably.

[0078] It is self-evident that enzymes having amino acid sequences in which some sequences are deleted, modified, substituted, or added are also included within the scope of the present application, as long as the amino acid sequence has the homology or identity mentioned in the present invention and exhibits an activity corresponding to the corresponding enzyme. That is, even if the present application describes "an enzyme having or including an amino acid sequence described by a specific sequence number," it is self-evident that enzymes having or including an amino acid sequence in which some sequences are deleted, modified, substituted, or added can also be used in the present application, as long as they have the same or corresponding activity as the enzyme consisting of the amino acid sequence of the corresponding sequence number.

[0079] (ii) In order to provide glucose to the enzymatic reaction of UDP-glucosyltransferase in step, at least one selected from the group consisting of UDP, UDP-glucose, and sucrose may be added, but is not limited thereto.

[0080] The reaction product of step (i) may be the reaction solution itself containing the produced steviol glycoside; or the steviol glycoside produced in step (i) separated and / or purified therefrom.

[0081] In the present invention, the reaction temperature of UDP-glucosyltransferase is not limited as long as its activity is exhibited, but may be 20 to 70°C, 25 to 65°C, 30 to 60°C, 35 to 55°C, 40 to 55°C, 45 to 55°C, 20 to 50°C, 25 to 45°C, 30 to 40°C, 50°C or 35°C.

[0082] In the present invention, the reaction pH of UDP-glucosyltransferase is not limited as long as its activity is exhibited, but may be pH 5 to 9, pH 5.5 to 8.5, pH 6 to 8, pH 6.5 to 7.5, or pH 7.2.

[0083] In the present invention, the reaction time of UDP-glucosyltransferase is not limited as long as the target steviol glycoside can be produced, but may be 1 to 72 hours, 3 to 60 hours, 3 to 48 hours, 6 to 36 hours, 9 to 24 hours, 12 to 24 hours, or 12 to 18 hours.

[0084] The production method of the present invention may additionally utilize sucrose synthase (SUS) in the reaction of step (ii), but is not limited thereto. The "sucrose synthase (SUS)" is an enzyme that produces fructose and UDP-glucose from sucrose and UDP. Therefore, when sucrose and UDP are supplied together with SUS in the reaction in which UDP-glucosyltransferase transfers glucose from UDP-glucose to steviol glycoside, SUS produces UDP-glucose from sucrose and UDP and provides it to UDP-glucosyltransferase (at this time, UDP-glucose may be supplied instead of or together with UDP). Therefore, using SUS in the reaction of the UDP-glucosyltransferase is more economical than supplying UDP-glucose separately. The above SUS can be provided on its own or in the form of a fusion protein with UDP-glucosyltransferase to produce steviol glycosides.

[0085] In one embodiment, the dextran sucrase comprises (i) dextran sucrase from Leuconostoc kimchii or (ii) dextran sucrase from Leuconostoc citreum and dextran sucrase from Leuconostoc lactis,

[0086] (i) The reaction product of step (i) includes a steviol glycoside (Rebaudioside D α') represented by the following chemical formula 2,

[0087] [Chemical Formula 2]

[0088]

[0089] Here,

[0090] R1 is And,

[0091] R2 is β-Glc-α-Glc (6→1),

[0092] UDP-glucosyltransferase includes UGT76G1,

[0093] Steviol glycosides may include, but are not limited to, steviol glycosides (Rebaudioside Mα or Rebaudioside Mα') represented by the following chemical formula 1:

[0094] [Chemical Formula 1]

[0095]

[0096] Here,

[0097] R1 is And,

[0098] R2 is This or;

[0099] R1 is And,

[0100] R2 is am.

[0101] In one embodiment, the dextran sucrase comprises a dextran sucrase derived from Leuconostoc citreum,

[0102] (i) The reaction product of step (i) comprises a steviol glycoside (Rebaudioside Aα) represented by the following chemical formula 2,

[0103] [Chemical Formula 2]

[0104]

[0105] Here,

[0106] R1 is And,

[0107] R2 is β-Glc,

[0108] UDP-glucosyltransferase includes UGT91D2,

[0109] The steviol glycoside may include, but is not limited to, a steviol glycoside (Rebaudioside Dα) represented by the following chemical formula 1.

[0110] [Chemical Formula 1]

[0111]

[0112] Here,

[0113] R1 is And,

[0114] R2 is am.

[0115] In one embodiment, the dextran sucrase comprises a dextran sucrase derived from Leuconostoc citreum,

[0116] (i) The reaction product of step (i) comprises a steviol glycoside (Rebaudioside Aα) represented by the following chemical formula 2,

[0117] [Chemical Formula 2]

[0118]

[0119] Here,

[0120] R1 is And,

[0121] R2 is β-Glc,

[0122] UDP-glucosyltransferase contains UGT76G1,

[0123] The steviol glycoside may include, but is not limited to, a steviol glycoside (Rebaudioside Dα(3)) represented by the following chemical formula 1.

[0124] [Chemical Formula 1]

[0125]

[0126] Here,

[0127] R1 is And,

[0128] R2 is am.

[0129] The production method of the present invention can utilize enzymes (dextran sucrase and UDP-glucosyltransferase) not only in purified form, but also in the form of a microorganism producing the enzyme; or a culture, culture supernatant, lysate, or extract thereof of the microorganism. That is, in the enzymatic reaction of each step, a substrate and a purified enzyme can be reacted, or a substrate and an enzyme-producing microorganism; or a culture, culture supernatant, lysate, or extract thereof of the microorganism (however, including the enzyme) can be reacted.

[0130] In the present invention, the culture means a medium containing an enzyme produced from a microorganism obtained by culturing an enzyme-producing microorganism in an appropriate medium, and may include the microorganism or may be in a cell-free form that does not contain the microorganism. The culture supernatant means a liquid obtained by removing solids from the culture, and contains the enzyme produced from the microorganism. The lysate includes a lysate obtained by lysing the cells of the microorganism or a supernatant obtained by centrifuging the lysate, and contains the enzyme produced from the microorganism. The extract is obtained by extracting the microorganism, its culture, culture supernatant, or lysate using an appropriate solvent or method, and contains the enzyme produced from the microorganism.

[0131] In this specification, unless otherwise stated, the microorganism according to the present invention means at least one selected from the group consisting of cells of the microorganism, freeze-dried cells of the microorganism, cultures of the microorganism, lysates of the microorganism, supernatants of the lysates, and extracts thereof.

[0132] The production method of the present invention may further comprise a step of recovering (e.g., separating and / or purifying) the steviol glycoside produced in step (i) prior to step (ii). Furthermore, the production method of the present invention may further comprise a step of recovering (e.g., separating and / or purifying) the target steviol glycoside after step (ii).

[0133] The above recovery can be performed using any suitable method known in the art, for example, centrifugation, filtration, precipitation, extraction, cell disruption, chromatography, or a combination thereof.

[0134] The novel Rebaudioside D and Rebaudioside M-like steviol glycosides according to the production method of the present invention exhibit improved water solubility, making them easy to apply to beverages and foods with high moisture content, and can greatly improve the industrial usability of steviol glycosides.

[0135] Figure 1a is a schematic diagram showing a production pathway of steviol glycosides using dextran sucrase and UGT91D2 according to one embodiment of the present invention.

[0136] Figure 1b is a schematic diagram showing a production pathway of steviol glycosides using dextran sucrase and UGT76G1 according to one embodiment of the present invention.

[0137] Figure 2 is a graph showing the HPLC analysis results of rebaduioside Aα, a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0138] Figure 3 is a graph showing the HPLC analysis results of rebaduioside Eα, a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0139] Figure 4 is a graph showing the HPLC analysis results of rebaduioside Dα, a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0140] Figure 5 is a graph showing the HPLC analysis results of rebaduioside Dα', a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0141] Figure 6 is a graph showing the HPLC analysis results of rebaudioside Mα, a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0142] Figure 7 is a graph showing the HPLC analysis results of rebaudioside Mα', a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0143] Figure 8 is a graph showing the HPLC analysis results of rebaudioside Dα(3), a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0144] Figure 9 is a graph showing the results of LC-ESI / MS analysis of rebaudioside Dα, a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0145] Figure 10 is a graph showing the results of LC-ESI / MS analysis of rebaudioside Mα, a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0146] Figure 11 is a graph showing the results of LC-ESI / MS analysis of rebaudioside Mα', a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0147] Figure 12 is a graph showing the results of LC-ESI / MS analysis of rebaudioside Dα(3), a product produced by a glucosyltransferase reaction according to one embodiment of the present invention.

[0148] Figures 13a to 13f are each a product of rebaudioside Dα produced by a glucosyltransferase reaction according to one embodiment of the present invention (Figure 13a). 1H NMR, (Fig. 13b) 13 C NMR, (Fig. 13c) 1 H- 1 H COSY, (Fig. 13d) TOCSY, (Fig. 13e) HSQC, (Fig. 13f) HMBC analysis results graphs.

[0149] Figures 14a to 14f are (Figure 14a) of rebaudioside M α, a product produced by a glucosyltransferase reaction according to one embodiment of the present invention. 1 H NMR, (Fig. 14b) 13 C NMR, (Fig. 14c) 1 H- 1 H COSY, (Fig. 14d) TOCSY, (Fig. 14e) HSQC, (Fig. 14f) HMBC analysis results graphs.

[0150] Figures 15a to 15f are (Figure 15a) of rebaudioside M α', a product produced by a glucosyltransferase reaction according to one embodiment of the present invention. 1 H NMR, (Fig. 15b) 13 C NMR, (Fig. 15c) 1 H- 1 H COSY, (Fig. 15d) TOCSY, (Fig. 15e) HSQC, (Fig. 15f) HMBC analysis results graphs.

[0151] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0152]

[0153] Example 1. Production of dextransucrase from Leuconostoc citreum KM20

[0154] To produce dextransucrase from Leuconostoc citreum KM20 (GenBank Accession No. ACA83218.1; SEQ ID NO: 1), polymerase chain reaction (PCR) was performed using KOD DNA polymerase (Toyobo, Osaka, Japan) using the genomic DNA of the Leuconostoc citreum KM20 strain screened from kimchi as a template and primers containing NcoI and XhoI restriction enzyme recognition sequences (forward: 5'-AAACCATGGAAAACGGCGAAGTGTGTCAGCG-3' (SEQ ID NO: 5) and reverse: 5'-TTTCTCGAGGTATGTTATTTTTTTCATTTCACC-3' (SEQ ID NO: 6)). The amplified dextransucrase gene was digested with restriction enzymes NcoI and XhoI and then inserted into the expression vector pET-23d(+) (Novagen, Darmstadt, Germany) with a C-terminal His-tag using the Gibson assembly method.

[0155] The prepared recombinant vector was transformed into Escherichia coli BL21 (DE3) codonPlus RIL (Stratagene, San Diego, CA, USA) and cultured in Luria-Bertani (LB) broth (10 mL) containing ampicillin (50 μg / mL) at 37°C until OD600 reached 0.5. Protein expression was induced by adding IPTG to a final concentration of 0.2 mM, and the culture was further cultured at 18°C ​​for 21 h.

[0156] Cultured cells were harvested by centrifugation at 8,000 Х g and suspended in a buffer containing 100 mM sodium phosphate buffer (pH 7.2), 10 mM imidazole, 1 mM PMSF, and a protease inhibitor cocktail (Roche, NJ, USA). After treatment with an ultrasonicator, the cells were centrifuged at 8,000 Х g for 30 min to obtain the supernatant.

[0157] The supernatant was applied to a Ni-Sepharose column (GE Healthcare, USA) and washed with 20 mM sodium phosphate buffer (pH 7.2) containing 300 mM NaCl and 20 mM imidazole, and the target protein was eluted from an elution buffer containing 300 mM imidazole in the same buffer.

[0158] The protein solution was then buffer-exchanged with 30 mM sodium acetate buffer (pH 5.5) and concentrated using an Amicon Ultra centrifugal concentrator (10,000 MWCO, Millipore, Billerica, MA, USA). The concentration of the purified protein was finally measured using the Bradford protein assay using BSA as a standard.

[0159]

[0160] Example 2. Production of Dextransucrase from Leuconostoc kimchiiIMSNU11154

[0161] To produce dextransucrase (GenBank Accession No. QBR47883.1; SEQ ID NO: 2) from Leuconostoc kimchii IMSNU11154, genomic DNA was isolated from Leuconostoc kimchii IMSNU11154 screened from kimchi using a Genomic Cell and Tissue mini kit (Nucleogen, Seoul, Korea). The isolated DNA was amplified using PrimeSTAR HS DNA polymerase (Takara, Tokyo, Japan). For amplification, primers designed based on the genome sequence of Leuconostoc kimchii IMSNU11154 (forward: 5'-GGCGAATTCGAATGAGAGATAAGAGAATAGT-3' (SEQ ID NO: 7), reverse: 5'-TAGCGGCCGCGTCAACGGCGTTACCGTCAT-3' (SEQ ID NO: 8)) were used, each of which included EcoRI and NotI restriction enzyme recognition sequences, and the stop codon was removed.

[0162] PCR was performed with initial denaturation at 94°C for 3 minutes, followed by 40 cycles of 98°C for 10 seconds, 52°C for 15 seconds, and 72°C for 5 minutes. The generated PCR products were purified using a gel extraction kit (Elpis-biotech, Daejeon, Korea) after 0.8% agarose gel electrophoresis of the target gene.

[0163] The purified gene was inserted into the pTOP Blunt V2 vector, transformed into E. coli DH5α, and cultured on LB solid medium containing ampicillin (70 μg / mL). Recombination was confirmed through colony PCR and DNA sequencing (Solgent, Daejeon, Korea). Subsequently, the gene digested with EcoRI and NotI was inserted into the same restriction enzyme sites of the pET-23d(+) vector to construct a recombinant plasmid containing a C-terminal 6xHis-tag.

[0164] The recombinant plasmid was transformed into E. coli BL21(DE3) CodonPlus RIL and cultured at 37°C in LB medium containing ampicillin (50 μg / mL) until OD600 reached 0.5, after which protein expression was induced at 18°C ​​for 21 h by adding IPTG at a concentration of 0.2 mM.

[0165] Cells were harvested by centrifugation (8,000 x g), suspended in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg / mL lysozyme, 1 mM DTT), and sonicated. The supernatant was loaded onto a HisTrap HP column (GE Healthcare, Madison, WI, USA), washed with 20 mM sodium phosphate buffer (pH 7.4), 25 mM imidazole, and 300 mM NaCl, and then the protein was eluted with an elution buffer containing 250 mM imidazole in the same buffer.

[0166] The eluted protein was dialyzed against 20 mM sodium acetate buffer (pH 5.2) and concentrated using an Amicon Ultra centrifugal concentrator (30 kDa MWCO, Millipore).

[0167]

[0168] Example 3. Production of Dextransucrase from Leuconostoc lactisEG001

[0169] To produce dextransucrase (GenBank Accession No. GQ213971; SEQ ID NO: 4) derived from Leuconostoc lactisEG001, a strain screened from kimchi was isolated from a mixed lactic acid bacteria culture broth derived from kimchi, and its ability to produce glucansucrase was confirmed. The gene was obtained through degenerate PCR and inverse PCR based on partial sequence information, and the entire gene was amplified using primers containing NcoI and NotI restriction enzyme recognition sequences (forward: 5'-ATCCATGGATAGTAACCAAAACACAACTGGTT-3' (SEQ ID NO: 9), reverse: 5'-ATGCGGCCGCCATATTGACGAGATCACCGTTG-3' (SEQ ID NO: 10)).

[0170] The PCR product was digested with NcoI and NotI and inserted into the pET-22b(+) expression vector (Novagen, USA) digested with the same restriction enzymes to produce a recombinant plasmid. This plasmid was transformed into E. coli BL21(DE3) and cultured at 37°C in LB medium containing 100 μg / mL ampicillin. When the OD600 reached 0.5, IPTG was added to a final concentration of 0.1 mM, and expression was induced at 20°C for 21 hours.

[0171] Cultured cells were suspended in a buffer containing 100 mM Tris-HCl (pH 7.4), 10 mM imidazole, 1 mM PMSF, and a protease inhibitor cocktail after centrifugation, and the cells were lysed using an ultrasonicator. The supernatant was centrifuged at 12,000 Х g for 15 min, reacted with Ni-NTA agarose (2 mL) at 4°C for 1 h, and then loaded onto a column.

[0172] The column was washed with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 20 mM imidazole, and the protein was eluted with the same buffer containing 250 mM imidazole. The eluted protein was dialyzed against a buffer containing 20% ​​(v / v) glycerol, 100 mM Tris-HCl (pH 7.4) and stored at 4°C.

[0173] The molecular weight of the purified EG001-derived glucansucrase was confirmed to be approximately 165 kDa through SDS-PAGE analysis, and the specific activity of the purified enzyme was approximately 2.4-fold increased compared to the crude enzyme. The final enzyme concentration was measured using the Bradford method.

[0174]

[0175] Experimental Example 1. HPLC Analysis of Reaction Products by Glucosyltransferase-Producing Enzyme

[0176] Analysis of the glycosylation products generated after the enzymatic reaction was performed using high-performance liquid chromatography (HPLC). The analysis was performed using a Shimadzu HPLC system (SCL-40 system) equipped with an LC-40D pump, an SPD-20A UV / Vis detector, a CTO-40C column heater, and LabSolutions software.

[0177] The sample was injected into a ZORBAX 300SB-C18 reversed-phase column (4.6 Х 150 mm, 5 μm; Agilent, Santa Clara, CA, USA) at an injection volume of 10 μL. The mobile phase consisted of acetonitrile (ACN) and water, with a gradient that increased linearly from 0% to 100% ACN over 60 min at a flow rate of 1.0 mL / min. The detection wavelength was set to 210 nm, and the column temperature was maintained at 30°C.

[0178]

[0179] Experimental Example 2. Production and Purification Method of Rebaudioside Aα

[0180] A 10 mL reaction solution containing 100 mM stevioside, 400 mM sucrose, and purified dextransucrase from Leuconostoc citreum KM20 in 20 mM sodium acetate buffer (pH 5.5) was prepared. The reaction was carried out at 30°C with stirring at 180 rpm for 48 h. The produced rebaudisodie Aα was confirmed by HPLC analysis (Fig. 2). After the reaction was completed, 66% (v / v) ethanol was added to the reaction solution to inactivate the enzyme, and the mixture was stored at -80°C overnight.

[0181] Afterwards, the reaction solution was centrifuged at 4°C and 7,010 Х g for 20 minutes, and the supernatant was concentrated under reduced pressure at 45°C using a rotary evaporator (Eylea N-1300, Eylea, Tokyo, Japan). The concentrated supernatant was loaded onto a Diaion HP-20 column (Tokyo, Japan), washed with water to remove residual sugars, and the glycosylation product was eluted using ethanol. The ethanol eluate was concentrated again using a rotary evaporator and redissolved in water.

[0182] The reconstituted sample was purified by medium pressure liquid chromatography (MPLC) using a Sfar C18 Duo reversed-phase column (240 g; Biotage, Uppsala, Sweden). MPLC was performed using a linear gradient starting with 100% water and gradually increasing the concentration to 50% (v / v) acetonitrile.

[0183]

[0184] Experimental Example 3. Production and Purification Method of Rebaudioside Eα

[0185] Enzymatic reactions were performed using a total of 10 mL of a reaction solution. The reaction solution contained 20 mM sodium acetate buffer (pH 5.5), 100 mM stevioside, 400 mM sucrose, and dextransucrase derived from Leuconostoc lactics EG001. The reaction was carried out for 48 h at 30°C with stirring at 180 rpm, and the produced Rebaudioside E α was confirmed by HPLC analysis (Fig. 3).

[0186] The reaction was terminated by adding 66% (v / v) ethanol to the reaction solution to inactivate the enzyme, and the reaction was completely stopped by storing the solution at -80°C overnight. Afterwards, the reaction solution was centrifuged at 4°C and 7,010 Х g for 20 minutes to separate the supernatant.

[0187] The separated supernatant was concentrated using a rotary evaporator under reduced pressure at 45°C, and the concentrated sample was loaded onto a Diaion HP-20 column. The column was washed with water to remove residual sugars, and the glycosylation product was eluted using ethanol. The fraction eluted with ethanol was concentrated again using a rotary evaporator and redissolved in distilled water. The redissolved sample was purified by MPLC (Medium Pressure Liquid Chromatography) using a Sfar C18 Duo reversed-phase column (240 g; Biotage, Uppsala, Sweden).

[0188]

[0189] Experimental Example 4. Production and Purification Method of Rebaudioside Dα

[0190] Rebaudioside Dα was produced using rebaudiodie Aα isolated from the reaction solution as an acceptor. The enzymatic reaction was performed in a 10 mL reaction solution containing 20 mM sodium phosphate buffer (pH 7.2), 4 mM UDP-G, 10 mM rebaudioside Aα, 5 mM EDTA, and UGT91D2. The reaction solution was stirred at 180 rpm at 35°C. 4 mM UDP-G was additionally added every 6 h after the start of the reaction, and the total reaction time was maintained for up to 18 h. The reaction was terminated by heat treatment at 100°C for 5 min. The produced rebaudioside Dα was confirmed by HPLC analysis (Fig. 4). After completion of the reaction, the reaction solution was centrifuged at 4°C and 7,010 Х g for 5 min, and the supernatant was analyzed by HPLC. Additionally, the supernatant was concentrated under reduced pressure at 45°C using a rotary evaporator, and the concentrated supernatant was purified through MPLC.

[0191]

[0192] Experimental Example 5. Production and Purification Method of Rebaudioside Dα'

[0193] For the production of rebaudioside Dα', a reaction mixture containing 20 mM stevioside, 40 mM sucrose, dextransucrase derived from Leuconostoc kimchiiIMSNU11154, and 20 mM sodium acetate buffer (pH 5.5) was prepared and reacted at 30°C for 24 hours. The product was analyzed by HPLC (Fig. 5).

[0194] Another method for producing Rebaudioside Dα' was to prepare a reaction mixture containing 20 mM stevioside, 40 mM sucrose, two kinds of dextransucrases derived from Leuconostoc citreum KM20 and Leuconostoc lactis EG001, and 20 mM sodium acetate buffer (pH 5.5), and react at 30°C for 24 hours.

[0195] The reaction was terminated by heating the reaction mixture for 5 minutes, after which three times the volume of cold ethanol was added to the reaction mixture. The mixture was stored at -80°C for 2 hours to precipitate dextran, and the precipitate was removed by centrifugation at 9,870 Х g for 20 minutes, and the supernatant was recovered.

[0196] The recovered supernatant was concentrated under reduced pressure using a rotary evaporator, and the concentrated sample was loaded onto Diaion HP-20 resin, washed with water at room temperature (25°C), and the derivative was eluted using 95% ethanol. The ethanol eluate was concentrated again using a rotary evaporator and fractionated using MPLC equipped with a Sfar Bio C18 column (300 Å, 20 μm, 240 g; Biotage).

[0197]

[0198] Experimental Example 6. Production and Purification Method of Rebaudioside Mα, Mα'

[0199] Rebaudioside Mα (RMα) and Rebaudioside Mα' (RMα') were produced using purified rebaudioside Dα' as an acceptor. The enzymatic reaction was performed by preparing a reaction solution containing 20 mM rebaudioside Dα', 40 mM sucrose, 0.3 mM UDP, 5 mM EDTA, and UGT76G1 in 20 mM sodium phosphate buffer (pH 7.2) and reacting at 50°C with stirring at 180 rpm for 12 h. The reaction was stopped by heating at 100°C for 5 min, and the produced RMα and RMα' were analyzed by HPLC, respectively (Figs. 6 and 7).

[0200] The enzyme reaction was terminated by heating the reaction mixture at 100°C for 5 minutes, and then 3 times the volume of chilled ethanol was added to the reaction solution to inactivate the enzyme. The mixture was stored at -80°C for 2 hours to precipitate dextran, and then centrifuged at 9,870 Х g for 20 minutes to remove the precipitate and separate the supernatant.

[0201] The separated supernatant was concentrated under reduced pressure using a rotary evaporator, and the concentrate was loaded onto Diaion HP-20 resin. The column was washed with water at room temperature (25°C) to remove residual sugars, and then eluted with 95% ethanol.

[0202] The eluted ethanol fraction was concentrated again using a rotary evaporator, and then fractional purification was performed using an MPLC system equipped with a Sfδr Bio C18 column (300 Å, 20 μm, 240 g; Biotage).

[0203]

[0204] Experimental Example 7. Production and Purification Method of Rebaudioside Dα(3)

[0205] Rebaudioside Dα(3) was produced using purified Rebaudioside Aα as an acceptor. The enzymatic reaction was performed in a total of 10 mL of reaction solution containing 20 mM sodium phosphate buffer (pH 7.2), 10 mM Rebaudioside Aα, 0.1 mM UDP, 5 mM EDTA, and UGT76G1 enzyme. The reaction was performed at 50°C with stirring at 180 rpm.

[0206] The reaction was stopped by heat treatment of the reaction solution at 100°C for 5 minutes. The production of Rebaudioside D α(3) was confirmed using HPLC (Fig. 8). The reaction solution was centrifuged at 4°C and 7,010 X g for 5 minutes, and the supernatant obtained therefrom was separated and purified using Diaion HP-20 and MPLC.

[0207]

[0208] Experimental Example 8. LC-ESI / MS Analysis of Reactants by Glucosyltransferase-Producing Enzymes

[0209] To confirm the structural characteristics of the purified steviol glycoside, analysis was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI / MS).

[0210] The analysis was performed using an ACQUITY Ultra-Performance Liquid Chromatography (UPLC) system from Waters (Waters, Milford, MA, USA). An ACQUITY UPLC HSS T3 column (100 Å, 1.8 μm, 2.1 × 50 mm; Waters Co., Ltd.) was used for separation. The mobile phase consisted of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B), and the separation was performed under the following linear gradient conditions:

[0211] · 0-2 minutes: 2% B

[0212] · 2-7 minutes: 2-35% B

[0213] · 7-13 minutes: 35-80% B

[0214] · 13-16.5 minutes: 80-100% B

[0215] The flow rate was 0.35 mL / min, the column temperature was maintained at 40°C, and the injection volume was 1 μL. Detection was performed using a Photodiode Array (PDA) detector.

[0216] Mass spectrometry was performed using a Xevo G2-XS Q-TOF system (Waters, Milford, MA, USA) in positive and negative ion modes using electrospray ionization (ESI). Ionization conditions were as follows:

[0217] · Capillary voltage: 2.5 kV

[0218] · Sample cone voltage: 40 V

[0219] · Collision energy was set to 6 eV under low energy conditions and 25-50 eV under high energy conditions.

[0220] · The mass scan range was set between m / z 60 and 1,500.

[0221] The molecular weight and structural characteristics of the glycosylation products were confirmed through LC-MS analysis performed under the conditions (Figs. 9 to 12).

[0222]

[0223] Experimental Example 9. NMR Analysis of Reactants by Glucosyltransferase-Producing Enzymes

[0224] Nuclear magnetic resonance (NMR) analysis was performed to elucidate the structure of the synthesized steviol glycosides. NMR analysis was performed at the Korea Basic Science Institute, Gwangju Center, using a UnityINOVA 500 and 600 MHz spectrometer from Varian (Walnut Creek, CA, USA).

[0225] The sample was dissolved in deuterated pyridine at a concentration of 20 mg / mL, transferred to a 5 mm diameter NMR tube, and analyzed. The structure of the synthesized steviol glycoside was 1 H-NMR and 13 This was confirmed through C-NMR analysis, and the binding positions between steviol aglycone and glucose residues were additionally identified through the following two-dimensional NMR technique:

[0226] ·Correlation spectroscopy (COSY),

[0227] ·Heteronuclear multiple bond correlation (HMBC),

[0228] ·Heteronuclear single quantum coherence (HSQC),

[0229] ·Total correlation spectroscopy (TOCSY),

[0230] ·HSQC-TOCSY.

[0231] The results are shown in Tables 1 to 6 and Figures 13 to 15.

[0232] rebaudioside Aα

[0233] [Table 1]

[0234]

[0235]

[0236] rebaudioside Eα

[0237] [Table 2]

[0238]

[0239]

[0240] rebaudioside D α

[0241] [Table 3]

[0242]

[0243]

[0244] rebaudioside D α'

[0245] [Table 4]

[0246]

[0247]

[0248] rebaudioside M α

[0249] [Table 5]

[0250]

[0251]

[0252]

[0253]

[0254] rebaudioside M α'

[0255] [Table 6]

[0256]

[0257]

[0258]

[0259]

[0260] The structure of steviol glycosides is shown in Table 7 below.

[0261] [Table 7]

[0262]

[0263]

[0264] Experimental Example 9. Temperature and pH stability of the reaction product by the enzyme producing glucosyltransferase.

[0265] To evaluate the thermal and pH stability of stevioside and various steviol glycoside derivatives (Rebaudioside A, D, M, Aα, Eα, Dα, Dα', Mα, Mα), each compound was prepared at a final concentration of 0.4% (w / v). Each sample was prepared by dissolving it in a solvent containing distilled water and DMSO in a 90:10 (v / v) ratio.

[0266] Thermal stability evaluation was performed by storing the prepared samples at 40°C, 80°C, and 120°C for 2 hours, and then calculating the relative stability based on the residual amount of each sample using high-performance liquid chromatography (HPLC) (Table 8).

[0267] For pH stability evaluation, samples were prepared under conditions adjusted from pH 1.4 to 10.0 using Britton-Robinson buffer, and after treatment at 80°C for 2 hours, stability was evaluated through HPLC analysis in the same manner (Table 9).

[0268] [Table 8]

[0269]

[0270] [Table 9]

[0271]

[0272]

[0273] Experimental Example 12. Evaluation of the Solubility of Reactants Using Glucosyltransferase-Producing Enzymes

[0274] To assess the water solubility of steviol glycosides, 1 mL of sterile deionized water was added to a 1.5 mL Eppendorf tube, followed by the addition of an excess of steviol glycoside sample. The sample mixture was stirred at 25°C for 24 hours to reach equilibrium.

[0275] After the reaction, the sample was centrifuged at 10,000 Х g for 20 minutes, and the supernatant was filtered into a new Eppendorf tube using a 0.2 μm hydrophilic PTFE syringe filter (Advantec Co. Ltd., Tokyo, Japan).

[0276] The filtered supernatant was frozen at -80°C for 2 hours, then freeze-dried (lyophilized) and the final residual mass of the steviol glycosides was measured to quantitatively evaluate the solubility (Table 10).

[0277] [Table 10]

[0278]

[0279]

[0280] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. Steviol glycoside represented by the following chemical formula 1: [Chemical Formula 1] Here, R1 is or And, R2 is , or am.

2. In paragraph 1, R1 is And, R2 is In, steviol glycosides.

3. In paragraph 1, R1 is And, R2 is In, steviol glycosides.

4. In paragraph 1, R1 is And, R2 is In, steviol glycosides.

5. In paragraph 1, R1 is And, R2 is In, steviol glycosides.

6. A sweetener composition comprising a steviol glycoside according to any one of claims 1 to 5.

7. A sweetener composition according to claim 6, wherein the steviol glycoside has increased water solubility compared to rebaudioside D or rebaudioside M. 8.(i) a step of reacting stevioside with dextran sucrase; a microorganism producing the same; or a culture, culture supernatant, lysate or extract thereof of the microorganism; and (ii) a step of reacting the reaction product of step (i) with UDP-glucosyltransferase; a microorganism producing the same; or a culture, culture supernatant, lysate or extract thereof of the microorganism; A method for producing a steviol glycoside, comprising:

9. In the 8th direction, Dextran sucrase includes (i) dextran sucrase derived from Leuconostoc kimchii or (ii) dextran sucrase derived from Leuconostoc citreum and dextran sucrase derived from Leuconostoc lactis, (i) The reaction product of step (i) comprises a steviol glycoside represented by the following chemical formula 2, [Chemical Formula 2] Here, R1 is And, R2 is β-Glc-α-Glc (6→1), UDP-glucosyltransferase includes UDP-glucosyltransferase 76G1 (UGT76G1), A method for producing a steviol glycoside comprising a steviol glycoside represented by the following chemical formula 1: [Chemical Formula 1] Here, R1 is And, R2 is This or; R1 is And, R2 is am.

10. In paragraph 8, Dextran sucrase contains dextran sucrase derived from Leuconostoc citreum, (i) The reaction product of step (i) comprises a steviol glycoside represented by the following chemical formula 2, [Chemical Formula 2] Here, R1 is And, R2 is β-Glc, UDP-glucosyltransferase includes UDP-glucosyltransferase 91D2 (UGT91D2), A method for producing a steviol glycoside comprising a steviol glycoside represented by the following chemical formula 1: [Chemical Formula 1] Here, R1 is And, R2 is am.

11. In paragraph 8, Dextran sucrase contains dextran sucrase derived from Leuconostoc citreum, (i) The reaction product of step (i) comprises a steviol glycoside represented by the following chemical formula 2, [Chemical Formula 2] Here, R1 is And, R2 is β-Glc, UDP-glucosyltransferase includes UDP-glucosyltransferase 76G1 (UGT76G1), A method for producing a steviol glycoside comprising a steviol glycoside represented by the following chemical formula 1: [Chemical Formula 1] Here, R1 is And, R2 is am.

12. In the 8th direction, (ii) A production method further comprising a step of recovering the steviol glycoside produced in step (i) prior to step (ii).

Images