Preparation method of a novel steviol glycoside derivative rebaudioside L2

Abstract

The present invention discloses a method for preparing a novel steviol glycoside derivative, rebaudioside L2, belonging to the field of biocatalytic synthesis. The present invention obtains a glycosyltransferase YjiC that catalyzes the monoglycosylation of rebaudioside A. The complete structure of the product is analyzed by liquid chromatography-mass spectrometry (LC-MS), 1D-NMR spectroscopy, and 2D NMR spectroscopy. The chemical structure of the obtained new product is 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester. The glycosyltransferase YjiC was coupled with the sucrose synthase AtSuSy from Arabidopsis thaliana to achieve efficient catalytic synthesis of rebaudioside L2 using rebaudioside A as a substrate. After 12 hours of reaction using 29.01 g / L (30 mmol / L) rebaudioside A as a substrate, 30.94 g / L of rebaudioside L2 was efficiently synthesized, and the yield of rebaudioside L2 reached 91.34%, providing a new, efficient and green route for the production of rebaudioside L2.

Description

Technical Field

[0001] The invention relates to a preparation method of a novel steviol glycoside derivative rebaudioside L2, belonging to the technical field of biocatalytic synthesis. Background Art

[0002] In recent years, the risk of dental caries, obesity, diabetes, hypertension, and cardiovascular disease has continued to increase worldwide, leading to a growing consumer demand for low- or zero-calorie sweeteners. Steviosides extracted from the stevia leaf are considered the most attractive sweeteners due to their high sweetness (50-450 times that of sucrose), lack of calories, and safety. Furthermore, steviosides have been found to possess important pharmacological activities, such as hypoglycemic and antihypertensive effects, diuretic effects, anti-inflammatory effects, anti-tumor effects, and immunomodulatory effects. Over 60 steviol glycosides have been identified from stevia, of which stevioside (5-10% of the leaf dry weight) and rebaudioside A (2-4% of the leaf dry weight) are the two most abundant components and the primary ingredients in commercially available steviol glycoside additives. Unfortunately, the lingering bitterness of steviol glycosides has limited their successful commercial application.

[0003] It has been found that the number and position of sugars attached to the C-13 and / or C-19 positions of steviol glycosides significantly influence sweetness and mouthfeel, but the specific relationship between structure and sweetness remains largely unexplained. A more effective approach to address this issue is to introduce a single glycosyl unit at different positions. Consequently, researchers have conducted numerous chemical and biological modifications of steviol glycosides to elucidate the structure-function relationship and, in the hope of obtaining steviol glycosides with enhanced sweetness quality. To date, a variety of enzymes have been reported for glycosylation of steviol glycosides, including cyclodextrin glycosyltransferases, glucanases, galactosidases, glucosidases, and fructosidases. However, these enzymes suffer from disadvantages such as low yields and mixed products, and they often introduce multiple glycosyl units simultaneously onto the substrate. In contrast, UDP-glycosyltransferases offer high conversion rates and regioselectivity. Therefore, exploring the potential of UDP-glycosyltransferases to achieve monoglycosylation of rebaudioside A at different positions is crucial for elucidating the relationship between steviol glycoside structure and sweetness quality. Summary of the Invention

[0004] To address these issues, the present invention explores the use of a glycosyltransferase, YjiC, from Bacillus sp., to catalyze the synthesis of the monoglycosylated derivative, rebaudioside L2, from rebaudioside A. This enzyme can be efficiently expressed in a soluble form in Escherichia coli and exhibits catalytic activity in the presence of uridine diphosphate glucose (UDPG). Furthermore, by constructing a UDPG recycling system, the efficient biosynthesis of rebaudioside L2 using E. coli lysate is achieved, providing an effective method for the industrial application of rebaudioside L2.

[0005] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0006] The first object of the present invention is to provide a compound, rebaudioside L2, the chemical structural formula of the compound, rebaudioside L2, is shown below:

[0007]

[0008] The second object of the present invention is to provide a recombinant bacterium expressing a glycosyltransferase, wherein the NCBI accession number of the amino acid sequence of the glycosyltransferase is WP_003232783.1.

[0009] In one embodiment, the recombinant bacterium further expresses sucrose synthase.

[0010] In one embodiment, the amino acid sequence of the sucrose synthase can be an amino acid sequence having sucrose synthase activity from any source.

[0011] In one embodiment, the NCBI accession number of the amino acid sequence of the sucrose synthase is NP_001031915.

[0012] In one embodiment, the recombinant bacteria uses Escherichia coli as a host cell.

[0013] The third object of the present invention is to provide a method for catalytic synthesis of rebaudioside L2, which comprises using UDP-glucose (UDPG) as a glycosyl donor and using a composition to catalyze rebaudioside A to prepare rebaudioside L2; the composition is one or more of glycosyltransferase YjiC or the above-mentioned recombinant bacteria or the cell lysate of the above-mentioned recombinant bacteria.

[0014] In one embodiment, the method uses rebaudioside A as a substrate and utilizes the cell lysate of the recombinant bacteria to perform a catalytic reaction.

[0015] In one embodiment, the cell lysate is the supernatant obtained by lysing the recombinant bacteria after induction of expression.

[0016] In one embodiment, the conditions of the catalytic reaction are: 5-50 mmol / L rebaudioside A, 50-800 mmol / L sucrose, 5-25% (v / v) DMSO, 100 mmol / L K2HPO4-KH2PO4 buffer, 100 mmol / L NaCl as the reaction system, glycosylation reaction at 20-45°C for 0-48 hours.

[0017] In one embodiment, the buffer has a pH of 5.5-9.0.

[0018] The present invention also provides the use of the above-mentioned glycosyltransferase YjiC or the above-mentioned recombinant bacteria or the above-mentioned method in preparing a product containing rebaudioside L2.

[0019] The fourth object of the present invention is to provide a sweetener containing the above-mentioned compound rebaudioside L2.

[0020] In one embodiment, the sweetener further contains a flavoring agent.

[0021] In one embodiment, the flavoring agent is one or more of ribose, xylose and xylitol, glucose, sorbitol, lactose, sucrose, palatinose, trehalose, maltodextrin or starch, lactic acid, malic acid and citric acid.

[0022] The present invention also provides the use of the compound rebaudioside L2 or the sweetener in the fields of food, medicine or chemical industry.

[0023] Beneficial effects:

[0024] (1) The present invention uses the nucleic acid sequence encoding glycosyltransferase YjiC to prepare a recombinant protein capable of catalyzing the glycosylation of rebaudioside A. The prepared recombinant protein can synthesize rebaudioside L2, a monoglycosylated derivative of rebaudioside A, using UDPG as a glycosyl donor and rebaudioside A as a substrate. This provides a new analogue for clarifying the relationship between the structure and sweetness of steviol glycosides.

[0025] (3) The present invention combines glycosyltransferase YjiC with sucrose synthase AtSuSy to construct a UDPG recycling system. By optimizing the coupled reaction system conditions, 30.94 g / L of rebaudioside L2 was synthesized from 29.01 g / L (30 mmol / L) of rebaudioside A with a high yield of 91.34%.

[0026] (4) The recombinant strain constructed in the present invention co-expresses a glycosyltransferase derived from Bacillus and a sucrose synthase derived from Arabidopsis. The cell lysate prepared after the recombinant strain is induced to express is used to catalyze the synthesis of rebaudioside L2 from rebaudioside A. There is no need to add a glycosyl donor or use an additional cell permeabilizer, which significantly reduces costs and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The biosynthetic pathway of rebaudioside A to rebaudioside L2 was shown to be catalyzed by glycosyltransferase YjiC.

[0028] Figure 2 This is the expression and purification analysis of the glycosyltransferase YjiC protein in Example 2.

[0029] Figure 3 This is a UPLC analysis of the synthesis of rebaudioside L2 from rebaudioside A catalyzed by glycosyltransferase YjiC in Example 3. Lane 1: Marker; Lane 2: Expression sample without IPTG induction; Lane 3: Crude enzyme solution; Lane 4: Crude enzyme solution supernatant; Lane 5: Crude enzyme solution precipitate; Lane 6: Purification flowthrough; Lane 7: Washed impurity protein sample; Lane 8: Eluted target protein sample.

[0030] Figure 4 This is the mass spectrometric analysis of rebaudioside L2, the glycosylation reaction product of rebaudioside A in Example 3.

[0031] Figure 5 This is the hydrogen spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside L2 in Example 4.

[0032] Figure 6 This is the carbon spectrum of the nuclear magnetic resonance spectroscopy analysis of the product rebaudioside L2 in Example 4.

[0033] Figure 7 This is the COSY spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside L2 in Example 4.

[0034] Figure 8 This is the TOCSY spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside L2 in Example 4.

[0035] Figure 9 This is the HSQC spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside L2 in Example 4.

[0036] Figure 10 This is the HMBC spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside L2 in Example 4.

[0037] Figure 11 This is the ROESY spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside L2 in Example 4.

[0038] Figure 12 This is the protein expression analysis of the lysate from the YjiC-AtSuSy glycosylation coupling reaction in Example 6. Lane 1: Marker; Lane 2: Expression sample without IPTG induction; Lane 3: Crude enzyme solution; Lane 4: Crude enzyme solution supernatant; Lane 5: Crude enzyme solution precipitate.

[0039] Figure 13 This is the effect of pH on the glycosylation coupling reaction buffer solution in Example 7.

[0040] Figure 14 This is the effect of temperature on the glycosylation coupling reaction in Example 8.

[0041] Figure 15 This is the effect of DMSO concentration on the glycosylation coupling reaction in Example 9.

[0042] Figure 16 This is the effect of sucrose concentration on the glycosylation coupling reaction in Example 10.

[0043] Figure 17 This is the effect of substrate concentration on the glycosylation coupling reaction in Example 11.

[0044] Figure 18 This is the effect of reaction time on the glycosylation coupling reaction in Example 12. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0047] The methods involved in the following embodiments are:

[0048] Glycosyltransferase Enzymatic Properties: Kinetic analysis of rebaudioside A by glycosyltransferase YjiC was performed in a 200 μL reaction system containing 5 mM UDPG, 10 mM MnCl2, 50 mM Bis-Tris pH 7.0, and 5 μg of purified protein sample. The rebaudioside A concentration ranged from 0.5 to 8 mM. The reaction was performed at 35°C for 20 minutes. The reaction was immediately quenched by heating at 95°C for 10 minutes. The sample was diluted with 5 volumes of methanol and centrifuged at 20,000 × g for 5 minutes to remove the protein precipitate. The supernatant was filtered through a 0.22 μm filter and analyzed by UPLC.

[0049] Determination of the yield of rebaudioside L2: Rebaudioside L2 standard solutions of different concentrations (1mM, 2mM, 3mM, 4mM, 5mM, 6mM) were prepared and analyzed by UPLC to obtain a rebaudioside L2 concentration standard curve equation of y = 4450596.9x + 346752.0, R 2= 0.99892. The yield of rebaudioside L2 was calculated based on the standard curve. Yield = actual yield of rebaudioside L2 / theoretical yield of rebaudioside L2.

[0050] Example 1 Acquisition of glycosyltransferase YjiC gene and construction of recombinant strain

[0051] The amino acid sequence (accession number: WP_003232783.1) and nucleic acid sequence (accession number: CP053102.1) of Bacillus glycosyltransferase were downloaded from Genbank, synthesized by Yixin Biotechnology Co., Ltd., and ligated into the multiple cloning restriction site of the vector pET-21b(+) to obtain the recombinant plasmid pET-21b(+)-YjiC.

[0052] The obtained plasmid pET-21b(+)-YjiC was sequenced and identified and transformed into Escherichia coli BL21(DE3) competent cells. Screening was performed on LB solid plates (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 20 g / L agar powder) containing 100 μg / mL ampicillin to obtain the recombinant strain E. coli BL2l(DE3)pET-21b(+)-YjiC.

[0053] Example 2 Induced expression of recombinant strains and purification of target protein

[0054] The recombinant strain E. coli BL21(DE3)pET-21b(+)-YjiC constructed in Example 1 was inoculated into 1 L 2×YT liquid medium (16 g / L peptone, 10 g / L yeast powder, 5 g / L NaCl) containing 100 μg / mL ampicillin and cultured at 135 rpm and 37°C until the OD 600 After the pH value was 0.6-0.8, the culture temperature was lowered to 18°C, and isopropyl-β-thiogalactoside (IPTG) was added at a final concentration of 0.1 mmol / L, and the culture was induced for 8 h.

[0055] The induced expression bacterial solution was centrifuged (7000 rpm, 7 min, 4°C), the supernatant discarded, and the cells collected. The cells were resuspended in lysis buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerol) at a ratio of 1 g of cells per 10 mL of lysis buffer. The cells were disrupted using a high-pressure homogenizer, and the disrupted bacterial solution was centrifuged (40,000 × g, 30 min). The supernatant was collected to obtain the crude enzyme solution.

[0056] The crude enzyme solution was treated with Ni +The column was purified by affinity chromatography. After the sample was loaded, 10 volumes of lysate were used to wash away the impurities and the target protein was eluted with elution buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 250 mmol / L imidazole, 10% glycerol). The eluted target protein was collected and desalted by a desalting column (HistrpTM 5 mL Desalting) with desalting buffer (25 mmol / L Tris-HCl, 150 mmol / L NaCl, 10% glycerol). After desalting, the protein was concentrated to 10 mg / mL and then subjected to subsequent reactions. The purified protein was detected by 10% SDS-PAGE gel electrophoresis. The results are shown in FIG. Figure 2 The target band was clear and the protein size was accurate. The K of YjiC to rebaudioside A was measured. m The value is 1.25±0.11mM, k cat The value is 0.71±0.02s -1 .

[0057] Example 3: Glycosylation reaction of rebaudioside A catalyzed by YjiC to synthesize rebaudioside L2

[0058] The purified glycosyltransferase YjiC obtained in Example 2 was used for glycosylation reaction ( Figure 1 ).

[0059] The glycosylation reaction was carried out in a 200 μL reaction system, and the reaction system was as follows: 50 mmol / L Tris-HCl pH 8.0, 5 mmol / L UDPG, 10 mmol / L MnCl2, 3 mmol / L rebaudioside A, and the concentration of the pure enzyme YjiC obtained in Example 2 was 10 μM. The reaction was carried out at 35 ° C for 20 min. After the reaction was completed, it was diluted with 5 volumes of methanol, centrifuged at 20,000 × g for 5 min, and filtered using a 0.22 μM filter membrane. The filtrate was subjected to ultra-performance liquid chromatography (UPLC) detection and analysis. UPLC used a BEH C18 1.7 μM reverse column from Waters, an injection volume of 2 μL, a column temperature of 40 ° C, a mobile phase using pipeline A: acetonitrile, pipeline B: 1.38 g / LNaH2PO4 buffer (pH 2.6), a flow rate of 0.3 mL / min, and the specific procedures are shown in Table 1:

[0060] Table 1 UPLC reaction procedure

[0061]

[0062]

[0063] Liquid phase analysis revealed that the results Figure 3As shown in the figure, compared with the rebaudioside A standard, it can be seen that a new product is obviously generated in the reaction system, and the reaction mixture is analyzed by mass spectrometry (MS) ( Figure 4 ), the negative ion mode results of LC-MS showed that there was a [MH] - The peak of the ion corresponds to the molecular formula C 50 H 80 O 28 , indicating that the product is a monosaccharide derivative of rebaudioside A, and there is a [M-Glc-H] - Since the ester bond at the C-19 position of stevioside is more easily broken and produces fragment ions in ESI-MS, it is speculated that a new glucose unit is added to the C-13-trisaccharide part of rebaudioside A.

[0064] Example 4 Structural Identification of Novel Monoglycosylated Derivatives of Rebaudioside A

[0065] Glycosyltransferase YjiC was used for large-scale (100 mL) glycosylation reaction to prepare novel derivatives. The reaction conditions were the same as in Example 3. Purification was performed using a semi-preparative HPLC system using a Shim-pack GIST C18 column (10×250 mm, 5 μm, SHIMADZU, Japan). The detection wavelength was 210 nm. The mobile phase was a mixture of acetonitrile and water. The solvent flow rate was 5 mL / min, the column temperature was 40°C, and the elution program was 0-35 min: 26% acetonitrile isocratic elution. 85 mg of high-purity product was obtained from the reaction mixture. Next, 1D ( 1 H and 13 C) and 2D NMR (COSY, TCOSY, HSQC, HMBC and ROESY) spectroscopy to analyze the complete structure of the product (see Figure 5-11 Data were collected using a Bruker Avance III 600 MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany). 1 The H spectrum detection frequency is 600MHz, 13 The C spectrum is at 151 MHz. 1H-NMR has typical steviol glycoside signal characteristics. Peaks with chemical shifts less than 2.5 ppm are derived from terpene aglycones, while peaks with chemical shifts between 3 and 6 ppm are derived from sugar rings.

[0066] from 1 H and 1 H- 13 C HSQC spectrum showed that H 6.07(δ C 95.56), δH 5.40(δ C 104.20),δ H 5.25(δ C 104.32), δ H 4.98(δ C 105.31),δ H 4.90(δ C 97.26) there are 5 abnormal protons, confirming that there are 5 sugar units in the structure of the product. H 6.07 (J = 8.4 Hz), δ H 5.40 (J = 7.3 Hz), δ H 5.25 (J = 7.9 Hz), δ H 4.98 (J = 7.9 Hz), δ H A high J dipole moment at 4.90 (J = 6.8 Hz) indicates that all five glucose residues are in a β-configuration. Detailed H and C chemical shift assignments of the new derivative were performed by 1D and 2D HMR, as shown in Table 2. The new product was identified as rebaudioside A, with a glucosyl group attached to the C-13 position of the diterpene core via a β-1,6 linkage. It was named rebaudioside L2. Its structural formula is 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester.

[0067] Table 2 1 H and 13 C Chemical Shift Assignment Table (pyridine-d5)

[0068]

[0069]

[0070] Example 5 Construction of glycosyltransferase and sucrose synthase recombinant plasmids and recombinant strains

[0071] The amino acid sequence (accession number: NP_001031915) and nucleic acid sequence (accession number: NM_001036838.2) of sucrose synthase AtSuSy from Arabidopsis thaliana were downloaded from Genbank, and E. coli-preferred codon optimization and gene synthesis were performed by Yixin Biotechnology Co., Ltd. The sucrose synthase encoding gene AtSuSy was ligated into the multiple cloning restriction site of pACYCDuet-1 to construct the recombinant plasmid pACYCDuet-1-AtSuSy. The resulting plasmid was sequenced and identified, and co-transformed with pET-21b(+)-YjiC into Escherichia coli BL21(DE3) competent cells. The cells were screened on LB solid plates (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, 20 g / L agar powder) containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol to obtain the recombinant strain E. coli BL21(DE3)YjiC-AtSuSy.

[0072] Example 6 Preparation of Cell Lysate for Glycosyltransferase and Sucrose Synthase Coupled Reaction

[0073] The recombinant strain E. coli BL21 (DE3) YjiC-AtSuSy constructed in Example 5 was inoculated into 5 L 2×YT medium supplemented with 100 μg / mL ampicillin and 34 μg / mL chloramphenicol, and cultured at 37°C and 135 rpm. 600 When the pH value was between 0.6 and 0.8, the temperature was lowered to 18°C and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM. Expression was continued at 18°C for 8 hours. The cells were then centrifuged at 7,000 × g for 7 minutes to collect the cells, washed three times with lysis buffer (100 mM K₂HPO₄-KH₂PO₄ (KPi) pH 8.0, 100 mM NaCl), and resuspended. The cells were disrupted using a high-pressure homogenizer. Immediately centrifuged at 40,000 × g for 30 minutes to remove cell debris. The supernatant was collected to obtain the coupled reaction cell lysate. Protein gel analysis was performed.

[0074] The results are as follows Figure 12 As shown, the results indicate that both glycosyltransferase and sucrose synthase are well expressed. Protein concentration in the cell lysate was measured using a Nano-Drop 2000 UV-Vis spectrophotometer. The prepared cell lysate was aliquoted and stored at -80°C or used directly in the coupling reaction.

[0075] Example 7 Effect of pH on the Glycosylation Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0076] The glycosylation coupling reaction system was placed in buffer solutions with different pH values to determine the effect of pH on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase. The selected buffer solutions were 100 mmol / L Bis-Tris pH 5.5-7.5, 100 mmol / L NaCl; 100 mmol / L KPi pH 5.5-8.0, 100 mmol / L NaCl; and 100 mmol / L Tris-HCl pH 7.5-9.0, 100 mmol / L NaCl.

[0077] The coupling reaction cell lysate was prepared as described in Example 6. The glycosylation coupling reaction system was 1 mL, which contained 40 mg / mL coupling reaction cell lysate, 30 mmol / L rebaudioside A, 200 mmol / L sucrose and 5% DMSO (v / v), and buffer; the reaction was carried out at 35°C for 6 hours. After the reaction was completed, 5 volumes of methanol were added to terminate the reaction, and then methanol was added to dilute it 6 times, centrifuged at 20,000 × g for 5 minutes, filtered through a 0.22 μM filter membrane, and then detected and analyzed by UPLC. The liquid phase detection method was carried out as described in Example 3, and the yield of rebaudioside L2 was calculated. The results showed that when the buffer was 100 mmol / L KPi pH 7.5 and 100 mmol / L NaCl, the yield of rebaudioside L2 could reach more than 55% ( Figure 13 ).

[0078] Example 8 Effect of Temperature on the Glycosylation Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0079] The glycosylation coupling reaction system was placed at different temperatures (20-45°C) to carry out the reaction, and the effect of temperature on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined.

[0080] The coupling reaction cell lysate was prepared as described in Example 6. The coupling reaction system was 1 mL, which contained 40 mg / mL coupling reaction cell lysate, 30 mmol / L rebaudioside A, 200 mmol / L sucrose, 10% DMSO (v / v) and 100 mmol / L KPi pH 7.5, 100 mmol / L NaCl, and the reaction was carried out for 6 hours. After the reaction was completed, 5 volumes of methanol were added to terminate the reaction, and then methanol was added to dilute 6 times, centrifuged at 20,000 × g for 5 minutes, filtered with a 0.22 μM filter membrane, and detected and analyzed by UPLC. The liquid phase detection method was carried out as described in Example 3, and the yield of rebaudioside L2 was calculated. The results showed that when the temperature was between 30 and 40 ° C, the yield of rebaudioside L2 could reach more than 50% ( Figure 14 ).

[0081] Example 9 Effect of DMSO Concentration on the Glycosylation Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0082] Different concentrations of DMSO (5%-25% (v / v)) were added to the glycosylation coupling reaction system to carry out the reaction, and the effect of DMSO concentration on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined.

[0083] The coupling reaction cell lysate was prepared as described in Example 6. The coupling reaction system was 1 mL, which contained 40 mg / mL coupling reaction cell lysate, 30 mmol / L rebaudioside A, 200 mmol / L sucrose, DMSO (5%-25% (v / v)) and 100 mmol / L KPi pH 7.5, 100 mmol / L NaCl, and the reaction was carried out at 35°C for 6 hours. After the reaction was completed, 5 volumes of methanol were added to terminate the reaction, and then the mixture was diluted 6 times, centrifuged at 20,000 × g for 5 minutes, filtered through a 0.22 μM filter membrane, and analyzed by UPLC. The liquid phase detection method was carried out as described in Example 3, and the yield of rebaudioside L2 was calculated. The results showed that when the DMSO concentration was 5% to 10% (v / v), the yield of rebaudioside L2 could reach more than 55% ( Figure 15 ).

[0084] Example 10 Effect of sucrose concentration on the coupled reaction of glycosyltransferase and sucrose synthase

[0085] Different concentrations of sucrose (50-800 mmol / L) were added to the glycosylation coupling reaction system to conduct the reaction, and the effect of sucrose concentration on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined.

[0086] The coupling reaction cell lysate was prepared as described in Example 6. The coupling reaction system was 1 mL, which contained 40 mg / mL coupling reaction cell lysate, 30 mmol / L rebaudioside A, sucrose (50-800 mmol / L), 10% (v / v) DMSO and 100 mmol / L KPi pH 7.5, 100 mmol / L NaCl, and the reaction was carried out at 35°C for 6 hours. After the reaction was completed, 5 volumes of methanol were added to terminate the reaction, and then the mixture was diluted 5 times, centrifuged at 20,000 × g for 5 minutes, filtered through a 0.22 μM filter membrane, and analyzed by UPLC. The liquid phase detection method was carried out as described in Example 3, and the yield of rebaudioside L2 was calculated. The results showed that when the sucrose concentration was 250-600 mmol / L, the yield of rebaudioside L2 could reach more than 60% ( Figure 16 ).

[0087] Example 11 Effect of Rebaudioside A Concentration on the Substrate of the Coupled Reaction of Glycosyltransferase and Sucrose Synthase

[0088] Different concentrations of rebaudioside A (5-50 mmol / L) were added to the glycosylation coupling reaction system to carry out the reaction, and the effect of sucrose concentration on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined.

[0089] The coupling reaction cell lysate was prepared as described in Example 6. The coupling reaction system was 1 mL, which contained 40 mg / mL coupling reaction cell lysate, rebaudioside A (5-50 mmol / L), 500 mmol / L sucrose, 10% (v / v) DMSO and 100 mmol / L KPi pH 7.5, 100 mmol / L NaCl, and the reaction was carried out at 35°C for 6 h. After the reaction was completed, 5 volumes of methanol were added to terminate the reaction, and then the reaction was diluted 5 times, centrifuged at 20,000 × g for 5 min, filtered with a 0.22 μM filter membrane, and analyzed by UPLC. The liquid phase detection method was carried out as described in Example 3, and the yield of rebaudioside L2 was calculated. The results showed that when the substrate concentration was 30 mmol / L, 24.00 g / L of rebaudioside L2 ( Figure 17 ).

[0090] Example 12 Effect of reaction time on the coupled reaction of glycosyltransferase and sucrose synthase

[0091] The glycosylation coupling reaction system was reacted for different times (0-24h) to determine the effect of sucrose concentration on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase.

[0092] The coupling reaction cell lysate was prepared as described in Example 6. The coupling reaction system was 20 mL, which contained 40 mg / mL coupling reaction cell lysate, 30 mmol / L rebaudioside A, 400 mmol / L sucrose, 10% (v / v) DMSO, 100 mmol / L KPi pH 7.5, and 100 mmol / L NaCl. The reaction was carried out at 35°C, and the reaction time was optimized to be 0-24 h. After the reaction was completed, 5 volumes of methanol were added to terminate the reaction, and then the mixture was diluted 5 times, centrifuged at 20,000 × g for 5 min, and filtered through a 0.22 μM filter membrane before detection and analysis by ultra-performance liquid chromatography (UPLC). The liquid phase detection method was carried out as described in Example 3, and the yield of rebaudioside L2 was calculated. The results showed that at the end of the reaction time of 12 h, 30.94 g / L rebaudioside L2 ( Figure 18 ).

[0093] Example 13 Sweetness Test of Rebaudioside L2

[0094] The sweetness test of Rebaudioside L2 was conducted using sucrose as a control. The sucrose sample was purchased from China National Pharmaceutical Group Co., Ltd. The purity of Rebaudioside L2 was 95%.

[0095] Three different concentrations of sucrose aqueous solutions were prepared at room temperature as controls: 1%, 5%, and 10% (w / v). 150 and 300 ppm rebaudioside L2 aqueous solutions were also prepared. Ten milliliters of each sample solution was placed in a 30-mL disposable tasting cup and subjected to a blind sweetness evaluation by nine trained and experienced volunteers. The evaluation results were the average of the scores given by the volunteers. In the evaluation, sweetness was measured on a scale of 10 based on a 10% sucrose aqueous solution (sweetness equal to 10% sucrose was 10 points, sweetness equal to 5% sucrose was 5 points, and so on). No sweetness was detected, resulting in a score of 0.

[0096] Blind taste tests showed that the sweetness scores of two different concentrations of rebaudioside L2 (150 and 300 ppm) were consistent, at 4.5 and 9.2, respectively. The results indicate that rebaudioside L2 is about 300 times sweeter than sucrose.

[0097] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for catalytically synthesizing rebaudioside L2, characterized in that: UDP-glucose is used as a glycosyl donor, and a composition is used to catalyze rebaudioside A to rebaudioside L2; the composition is one or more of glycosyltransferase YjiC, a recombinant bacterium expressing glycosyltransferase YjiC and sucrose synthase, or a cell lysate of the recombinant bacterium expressing glycosyltransferase YjiC and sucrose synthase; wherein the reaction conditions are as follows: a coupling reaction system of 20 mL, containing 40 mg / mL of the composition, 30 mmol / L rebaudioside A, 400 mmol / L sucrose, 10% DMSO by volume, 100 mmol / L KPi at pH 7.5, and 100 mmol / L NaCl, reacting at 35°C for 12 h; after the reaction is completed, 5 volumes of methanol are added to terminate the reaction; The NCBI accession number of the amino acid sequence of the glycosyltransferase YjiC is WP_003232783.1; The chemical structural formula of the rebaudioside L2 is as follows: 。 2. The method according to claim 1, characterized in that The cell lysate is the supernatant obtained by lysing the recombinant bacteria expressing glycosyltransferase YjiC and sucrose synthase after induction of expression.

3. The method according to claim 1 or 2, characterized in that The recombinant bacteria uses Escherichia coli as a host cell.

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