A preparation method of a novel steviol glycoside derivative rebaudioside M8

Abstract

The application discloses a preparation method of a novel steviol glycoside derivative rebaudioside M8 and belongs to the field of biological catalysis synthesis. The application obtains a glycosyltransferase UGT94E13 with catalytic rebaudioside D monosaccharide glycosylation activity through excavation, analyzes the complete structure of a product through LC-MS, 1D and 2D NMR spectrum, and obtains a novel steviol glycoside, rebaudioside M8, which is a rebaudioside D monosaccharide glycosylation derivative. The application constructs a coupling reaction of the glycosyltransferase UGT94E13 and a sucrose synthase AtSuSy, realizes efficient catalytic synthesis of rebaudioside M8 with rebaudioside D as a substrate, and efficiently synthesizes 5.71 g / L of rebaudioside M8, with a rebaudioside M8 yield of 88.52%, thereby providing a new efficient and green way for rebaudioside M8 production.

Description

Technical Field

[0001] This invention relates to a method for preparing a novel steviol glycoside derivative, rebaudioside M8, which belongs to the field of biocatalytic synthesis technology. Background Technology

[0002] In recent years, the risk of tooth decay, obesity, diabetes, hypertension, and cardiovascular disease has been increasing worldwide, leading to a growing consumer demand for low-calorie or calorie-free sweeteners. Steviosides, extracted from stevia, are considered the most attractive sweeteners due to their high sweetness (50-450 times that of sucrose), calorie-free nature, and safety. Furthermore, steviosides have been found to possess important pharmacological activities, such as hypoglycemic, hypotensive, diuretic, anti-inflammatory, antitumor, and immunomodulatory effects. Currently, over sixty steviosides have been identified in stevia, with steviol (5-10% of the leaf's dry weight) and rebaudioside A (2-4% of the leaf's dry weight) being the two most abundant components and the main ingredients in commercially available stevioside additives. Unfortunately, consuming steviosides leaves a lingering bitter taste, which limits their more successful commercial application.

[0003] It has been found that the number and position of sugars linked at the C-13 and / or C-19 positions of steviol glycosides significantly affect sweetness and mouthfeel, but the specific relationship between structure and sweetness remains insufficiently elucidated. A relatively effective approach to address this issue is to introduce a single glycosyl unit at different positions. Therefore, researchers have conducted numerous chemical and biological modifications of steviol glycosides to elucidate the relationship between structure and function and to obtain steviol glycosides with superior sweetness. To date, various enzymes have been reported for the glycosylation modification of steviol glycosides, such as cyclodextrin glycosyltransferases, glucanases, galactosidases, glucosidases, and fructosidases. However, these enzymes suffer from drawbacks such as low yields and mixed products, and often introduce multiple glycosyl units onto the substrate simultaneously. In contrast, UDP-glycosyltransferases exhibit high conversion rates and regioselectivity. Therefore, exploring UDP-glycosyltransferases to achieve monosaccharidation of rebaudioside D at different positions is of great significance for elucidating the relationship between steviol glycoside structure and sweetness quality. Summary of the Invention

[0004] To address the aforementioned issues, this invention discovers a glycoside transferase, UGT94E13, derived from Gardenia jasminoides, that catalyzes the synthesis of the monosaccharide derivative rebaudioside M8 from rebaudioside D. This enzyme can be expressed efficiently and solublely in Escherichia coli and exhibits catalytic activity in the synthesis of rebaudioside M8 from rebaudioside D in the presence of uridine diphosphate glucose (UDPG). Furthermore, by constructing a UDPG recycling system, rebaudioside M8 is efficiently biosynthesized using E. coli lysate, providing an effective method for the industrial application of rebaudioside M8.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] The first object of the present invention is to provide a compound having the chemical structure 13-[(2-O- β -D-glucopyranosyl-3-O-(6-O- β -D-glucopyranosyl)- β -D-glucopyranosyl- β -D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(2-O- β -D-glucopyranosyl- β [-D-glucopyranosyl) ester], the chemical structure of which is shown below:

[0007]

[0008] A second object of the present invention is to provide a sweetener composition containing the compound.

[0009] In one embodiment, the sweetener composition further comprises one or more of rebaudine A, rebaudine D, rebaudine E, rebaudine O, inulin, β-glucan, mogroside, xylitol, erythritol, maltitol, sucralose, aspartame, acesulfame K, or neotame.

[0010] In one embodiment, the sweetener composition further contains a flavoring agent.

[0011] In one embodiment, the flavoring agent is one or more of ribose, xylose and xylitol, glucose, sorbitol, lactose, sucrose, palaginose, trehalose, maltodextrin, or starch, lactic acid, malic acid, and citric acid.

[0012] A third object of the present invention is to provide the use of the compound or the sweetener composition in the preparation of pharmaceuticals.

[0013] A fourth object of the present invention is to provide the use of the compound or the sweetener composition in the preparation of sweeteners.

[0014] A fifth objective of this invention is to provide a recombinant bacterium for synthesizing the compound, wherein the recombinant bacterium overexpresses glycosyltransferase UGT94E13, the amino acid sequence of which has the NCBI accession number MN944055.1.

[0015] In one embodiment, the nucleotide sequence of the glycosyltransferase UGT94E13 has the NCBI accession number ARU08119.1.

[0016] In one embodiment, the recombinant bacteria also express sucrose synthase.

[0017] In one embodiment, the amino acid sequence of the sucrose synthase can be any amino acid sequence with sucrose synthase activity from any source.

[0018] In one embodiment, the NCBI accession number for the amino acid sequence of the sucrose synthase is NP_001031915.

[0019] In one embodiment, the nucleotide sequence of the sucrose synthase is shown in SEQ ID NO.1.

[0020] In one embodiment, the recombinant bacteria uses Escherichia coli as the host cell.

[0021] In one embodiment, the recombinant bacteria uses the pET series as an expression vector.

[0022] In one embodiment, the recombinant bacteria uses pET-21b(+) as an expression vector.

[0023] A third object of the present invention is to provide a method for catalytic synthesis of said compound, said method comprising (a) or (b):

[0024] (a) Using UDP-glucose as a glycosyl donor and rebaudioside D as a substrate, the reaction was catalyzed by glycosyltransferase UGT94E13 and sucrose synthase.

[0025] (b) The recombinant bacteria were fermented and cultured, the bacterial culture was collected, the supernatant was collected after rupture, and the supernatant was used to carry out the catalytic reaction with UDP-glucose as glycosyl donor and rebaudioside D as substrate.

[0026] In one embodiment, the catalytic reaction conditions are as follows: using 2-10 mmol / L rebaudioside D, 0-800 mmol / L sucrose, 5-25% (v / v) DMSO, 80-120 mmol / L K2HPO4-KH2PO4 buffer, and 80-120 mmol / L NaCl as the reaction system, the glycosylation reaction is carried out at 20-50°C for 0-48 h.

[0027] In one embodiment, the buffer solution has a pH of 5.5-9.0.

[0028] The present invention also provides the use of glycosyltransferase UGT94E13 or the recombinant bacteria or the method in the preparation of the compound or a product containing the compound.

[0029] Beneficial effects:

[0030] This invention uses UDPG as a glycosyl donor and rebaudioside D as a substrate to catalyze the synthesis of a novel steviol glycoside—rebaudioside D monosaccharide derivative rebaudioside M8—using glycosyltransferase UGT94E13, providing a new sweetener. It also provides a new analogue for elucidating the relationship between steviol glycoside structure and sweetness. Furthermore, this invention combines glycosyltransferase UGT94E13 with sucrase synthase... At A UDPG recycling system was constructed using SuSy in combination. Through optimization of the coupling reaction conditions, 5.71 g / L rebaudin M8 was synthesized from 5.64 g / L (5 mmol / L) rebaudin D with a high yield of 88.52%. The recombinant strain constructed in this invention co-expresses a glycosyltransferase from gardenia and a sucrose synthase from Arabidopsis. The cell lysate prepared after induction of expression by the recombinant strain catalyzes the synthesis of rebaudin M8 from rebaudin D, eliminating the need for glycosyl donors and additional cell permeabilizers, significantly reducing costs and being environmentally friendly. Attached Figure Description

[0031] Figure 1 This demonstrates the biosynthetic pathway of rebaudioside M8 catalyzed by glycosyltransferase UGT94E13 to produce rebaudioside M8 from rebaudioside D.

[0032] Figure 2 This section describes the expression and purification analysis of the glycosyltransferase UGT94E13 protein in Example 2. Lane 1: Marker; Lane 2: Sample without IPTG induction; Lane 3: Crude enzyme solution; Lane 4: Crude enzyme solution supernatant; Lane 5: Crude enzyme solution precipitate; Lane 6: Purification permeate; Lane 7: Washed sample containing other proteins; Lane 8: Eluted sample containing the target protein.

[0033] Figure 3This is a UPLC analysis chromatogram of the synthesis of rebaudioside M8 from rebaudioside D catalyzed by glycosyltransferase UGT94E13 in Example 3.

[0034] Figure 4 Mass spectrometry analysis of rebaudioside M8, the product of the rebaudioside D glycosylation reaction in Example 3.

[0035] Figure 5 The proton NMR spectrum of the product rebaudioside M8 in Example 4 is shown.

[0036] Figure 6 The carbon NMR spectrum of lebodiin M8, the product in Example 4.

[0037] Figure 7 The COSY spectrum is the nuclear magnetic resonance spectrum of lebodiin M8, the product in Example 4.

[0038] Figure 8 The TOCSY spectrum is obtained from the nuclear magnetic resonance spectroscopy analysis of the product lebodiin M8 in Example 4.

[0039] Figure 9 The HSQC spectrum of the product lebodiin M8 in Example 4 is shown.

[0040] Figure 10 The HMBC spectrum of the product lebodiin M8 in Example 4 is obtained by nuclear magnetic resonance spectroscopy analysis.

[0041] Figure 11 The ROESY spectrum is obtained from the nuclear magnetic resonance spectroscopy analysis of the product lebodiin M8 in Example 4.

[0042] Figure 12 For example, UGT94E13- in Example 6 At Analysis of protein expression in SuSy glycosylation coupling reaction lysate. Lane 1: Marker; Lane 2: Sample without IPTG induction; Lane 3: Crude enzyme solution; Lane 4: Crude enzyme solution supernatant; Lane 5: Crude enzyme solution precipitate.

[0043] Figure 13 This illustrates the effect of pH on the glycosylation coupling reaction buffer solution in Example 7.

[0044] Figure 14 This illustrates the effect of temperature on the glycosylation coupling reaction in Example 8.

[0045] Figure 15 The effect of DMSO concentration on the glycosylation coupling reaction in Example 9.

[0046] Figure 16 The effect of sucrose concentration on the glycosylation coupling reaction in Example 10.

[0047] Figure 17 The effect of reaction time on the glycosylation coupling reaction in Example 11. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0050] The culture media involved in the following examples:

[0051] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder.

[0052] 2×YT liquid medium: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl.

[0053] The methods involved in the following embodiments:

[0054] Determination of the enzymatic properties of glycosyltransferase: The kinetic analysis of rebaudioside D by glycosyltransferase UGT94E13 was performed in a 200 μL reaction system containing 5 mM UDPG, 10 mM MnCl2, 50 mM Tris (pH 8.0), and 5 μg of purified protein sample (glycosyltransferase UGT94E13). The concentration of rebaudioside D was 0–0.5 mM. The reaction temperature was 35 °C, and the reaction time was 2 h. The reaction mixture was reacted at 35 °C for 2 h, then quenched by heating at 95 °C for 5 min, and the reaction mixture was diluted with two volumes of methanol. Centrifugation at 20,000 × g for 5 min was performed to remove precipitates. The supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis.

[0055] Enzyme activity is defined as the amount of enzyme required to synthesize 1 μM rebaudioside M8 within 1 hour.

[0056] Determination of the yield of rebaudioside M8: The glycosylated product was dissolved in dimethyl sulfoxide to prepare a 5 mM mother liquor. The mother liquor was then diluted with methanol to prepare solutions of various concentrations: 0, 0.1, 0.25, 0.5, 0.75, and 1 mM. After filtration through a 0.22 μM filter membrane, the standard solutions were analyzed by UPLC. The standard curve equation for the rebaudioside M8 concentration was obtained as y = 2601014.39560x - 3188.57143, R0. 2=0.99877. The yield of rebaudioside M8 was calculated based on the standard curve. Yield = Actual yield of rebaudioside M8 / Theoretical yield of rebaudioside M8.

[0057] Waters Acquity UPLC system: BEH C18 1.7μM column (2.1×50 mm), HPLC conditions: organic phase – acetonitrile, aqueous phase – ultrapure water; flow rate 0.3 mL / min; column temperature 40℃; UV detection wavelength 210 nm; detection program: 0-1 min 15% organic phase, 6 min 40% organic phase, 7-8 min 15% organic phase.

[0058] Example 1: Obtaining the glycosyltransferase UGT94E13 gene and constructing recombinant strains

[0059] The amino acid sequence (accession number: MN944055.1) and nucleotide sequence (accession number: ARU08119.1) of gardenia glycosyltransferase were downloaded from Genbank. The gene was synthesized by Yixin Biotechnology Co., Ltd. and ligated into the polyclonal restriction site of the vector pET-21b(+) to obtain the recombinant plasmid pET-21b(+)-UGT94E13.

[0060] The resulting plasmid pET-21b(+)-UGT94E13 was sequenced, identified, and transformed into E. coli. E. coli Recombinant strains were obtained by screening BL21(DE3) competent cells using LB agar plates containing 100 μg / mL ampicillin. E. coli BL21(DE3)pET-21b(+)-UGT94E13.

[0061] Example 2: Induction of recombinant strain expression and purification of target protein

[0062] The recombinant strain constructed in Example 1 E. coli BL21(DE3) pET-21b(+)-UGT94E13 was inoculated into 1 L of 2×YT liquid medium containing 100 μg / mL ampicillin and cultured at 135 rpm and 37°C until OD. 600 After the concentration was reduced to 0.6-0.8, the culture temperature was lowered to 18℃, and isopropyl-β-thiogalactoside (IPTG) was added to a final concentration of 0.1 mmol / L for induced culture for 8 h.

[0063] Centrifuge the induced bacterial culture (7000 rpm, 7 min, 4℃), discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells 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 bacterial cells per 10 mL of lysis buffer. Disrupt the culture using a high-pressure homogenizer, then centrifuge the disrupted culture (40000×g, 30 min), and collect the supernatant to obtain the crude enzyme solution.

[0064] The crude enzyme solution was utilized with Ni + Affinity chromatography purification was performed using a column. After sample loading, contaminating proteins were washed with 10-fold volume of lysis buffer, followed by elution with elution buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 250 mmol / L imidazole, 10% glycerol). The eluted protein was collected and desalted using a Histrup™ 5 mL desalting column with desalting buffer (25 mmol / L Tris-HCl, 150 mmol / L NaCl, 10% glycerol). The desalted protein was concentrated to 10 mg / mL for subsequent reactions. The purified protein was analyzed by 10% SDS-PAGE gel electrophoresis. The results are shown in the figure. Figure 2 A purified enzyme with a clear target band and accurate protein size was successfully obtained. The effect of UGT94E13 on lebaudine D was measured. K m The value was 0.89 ± 0.05 mM. k cat The value was 0.33 ± 0.08 min. -1 The enzyme activity of the pure enzyme UGT94E13 was measured to be 360 ​​mU / mg.

[0065] Example 3: Glycosylation reaction of rebaudioside D catalyzed by UGT94E13 to synthesize rebaudioside M8

[0066] The purified glycosyltransferase UGT94E13 obtained in Example 2 was used for glycosylation reaction ( Figure 1 ).

[0067] The glycosylation reaction was carried out in a 200 μL reaction system as follows: 50 mmol / L Tris pH 8.0, 5 mmol / L UDPG, 10 mmol / L MnCl2, 0.5 mmol / L rebaudioside D, and the purified enzyme UGT94E13 obtained in Example 2 was at a concentration of 5 μM. The reaction was carried out at 35 °C for 4 h. The reaction was then quenched by heating at 95 °C for 5 min, and the reaction mixture was diluted with 2 volumes of methanol. The mixture was centrifuged at 20000 × g for 5 min to remove the precipitate. The supernatant was filtered through a 0.22 μm filter membrane and used for UPLC and LC-MS analysis. The Waters Acquity UPLC system used a BEH C18 1.7 μM column (2.1 × 50 mm) with the following HPLC conditions: organic phase - acetonitrile, aqueous phase - ultrapure water; flow rate 0.3 mL / min; column temperature 40℃; UV detection wavelength 210 nm; detection program: 0-1 min 15% organic phase, 6 min 40% organic phase, 7-8 min 15% organic phase.

[0068] The results can be obtained through liquid phase analysis, as follows: Figure 3 As shown, compared with the standard of rebaudioside D, it can be seen that a significant new product was formed in the reaction system, and the reaction mixture was analyzed by mass spectrometry (MS). Figure 4 The negative ion mode results of LC-MS showed that there is a [M] at m / z 1289.5421. H] The peak of the ion corresponds to the molecular formula C. 56 H 90 O 33 This indicates that the product is a monosaccharide derivative of rebaudioside D.

[0069] Example 4: Structural Identification of Novel Rebaudioside D Monosaccharide Derivatives

[0070] Novel derivatives were prepared by using glycosyltransferase UGT94E13 for large-scale (100 mL) glycosylation reaction. The reaction system was as follows: 2 mM Reb D, 10 μM glycosyltransferase, 5 mM UDPG, 10 mM MnCl2, 50 mM Tris, pH 8.0. The reaction mixture was reacted at 35℃ for 24 h, then quenched by heating at 95℃ for 5 min, and centrifuged at 20000×g for 5 min to remove precipitate. The supernatant was filtered through a 0.22 μm filter membrane and purified using a semi-preparative high-performance liquid chromatography (HPLC) system. The system used a Shim-pack GIST C18 column (10×250 mm, 5 µm, SHIMADZU, Japan). The HPLC conditions were: organic phase – acetonitrile, aqueous phase – ultrapure water; flow rate 5 mL / min; time program: 0–28 min 23% organic phase; 28.5–30.5 min 60% organic phase; 31–35 min 23% organic phase. Column temperature: 40℃; UV detection wavelength 210 nm. The obtained sample was dissolved in deuterated pyridine and purified by 1D (… 1 H and 13 The complete structure of the product was analyzed using C) and 2D NMR (COSY, TCOSY, HSQC, HMBC, and ROESY) spectra. Data were collected using a Bruker Avance III 600 MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany). 1 The H-spectrum detection frequency is 600 MHz. 13 The C-spectrum was 151 MHz. The 1H-NMR spectrum exhibited typical steviol glycoside signal characteristics. Peaks with chemical shifts less than 2.8 ppm originated from terpene aglycones, while peaks with chemical shifts from 3.6 to 6.3 ppm originated from the sugar ring.

[0071] from 1 H and 1 H- 13 C HSQC spectra show δ H 6.29 (δ) C 93.45), δ H 5.53 (δ) C 104.28), δ H 5.46 (δ) C 105.43), δ H 5.25 (δ) C 104.37), δ H 5.06 (δ) C 97.38), δ H 4.81 (δ) CThe presence of 6 aberrant protons (104.26) confirms the presence of 6 sugar units in the product's structure. Simultaneously, in the δ... H 6.29 (7.3 Hz), δ H 5.53 (7.8 Hz), δ H 5.46 (7.7 Hz), δ H 5.25 (7.9 Hz), δ H 5.06 (7.5 Hz), δ H A high Hz was observed at 4.81 (7.7 Hz). J The dipole moment indicates that all 6 glucose residues are β Configuration. The chemical shifts of H and C in the new derivatives were assigned in detail by 1D and 2D HMR. Figures 5-11 As shown in Table 1, the new product was determined to be derived from the glucose group at the C-13 position of the core of rebaudioside D diterpene via... β A glucose sac is linked by a -1,6- bond, which is named lebaudine M8. Its structural formula is 13-[(2-O- β -D-glucopyranosyl-3-O-(6-O- β -D-glucopyranosyl)- β -D-glucopyranosyl- β -D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(2-O- β -D-glucopyranosyl- β -D-glucopyranosyl) ester].

[0072] Table 1 1 H and 13 C chemical shift assignment table (pyridine- d 5)

[0073]

[0074] Example 5 Construction of recombinant plasmids and recombinant strains of glycosyltransferase and sucrose synthase

[0075] Download sucrose synthase from Arabidopsis thaliana from Genbank. At The amino acid sequence of SuSy (accession number: NP_001031915) and its nucleic acid sequence (accession number: NM_001036838.2) were optimized for E. coli codon preference and synthesized by Yixin Biotechnology Co., Ltd. The gene encoding sucrose synthase will be used in this process. AtSuSy was ligated to the multiple cloning restriction site of pACYCDuet-1 to construct the recombinant plasmid pACYCDuet-1- At SuSy sequenced and identified the obtained plasmid, and compared it with pET-21b(+)- UGT94E13 Co-transformed into Escherichia coli E. coli Recombinant strains were obtained by screening BL21(DE3) competent cells using LB agar plates containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. E. coli BL21(DE3)UGT94E13- At SuSy.

[0076] Example 6 Preparation of cell lysate from the coupling reaction of glycosyltransferase and sucrose synthase

[0077] The recombinant strain constructed in Example 5 E. coli BL21 (DE3) UGT94E13-AtSuSy was inoculated into 5 L of 2×YT medium supplemented with 100 μg / mL ampicillin and 34 μg / mL chloramphenicol, and cultured at 37°C with shaking at 135 rpm. The culture was continued until the bacterial cells reached the OD value. 600 When the pH value reached 0.6-0.8, the temperature was lowered to 18℃, and IPTG was added to a final concentration of 0.2 mM. Expression was induced at 18℃ for another 8 h. The cells were then collected by centrifugation at 7000×g for 7 min. The cells were washed three times with lysis buffer (100 mM K₂HPO₄-KH₂PO₄(KPi) pH 8.0, 100 mM NaCl) and resuspended. The cells were homogenized using a high-pressure homogenizer. Immediately afterward, the cells were centrifuged at 40000×g for 30 min to remove cell debris. The supernatant was collected as the coupled-reaction cell lysate, and protein gel analysis was performed.

[0078] The results are as follows Figure 12 As shown in the results, both glycosyltransferase and sucrase synthase were well expressed. Protein concentrations in the cell lysates were determined using a Nano-Drop 2000 UV-Vis spectrophotometer. The prepared cell lysates were aliquoted and stored at -80°C, or used directly as crude enzyme solutions for coupling reactions.

[0079] Example 7: Effect of pH on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase

[0080] 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 mM KPi pH 5.5-8.0 (containing 100 mM NaCl) and 100 mM Tris pH 7.0-9.0 (containing 100 mM NaCl).

[0081] Cell lysate for the coupling reaction was prepared as described in Example 6. The glycosylation coupling reaction system consisted of 1 mL containing 40 mg / mL crude enzyme, 5 mM Reb D, 200 mM sucrose, and 5% dimethyl sulfoxide (v / v). The reaction mixture was reacted at 35°C for 3 h; then the reaction was quenched by heating at 95°C for 5 min, and the reaction mixture was diluted with 4 times its volume of methanol; centrifuged at 20000×g for 5 min to remove precipitate; the supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis. The liquid chromatography detection method was performed as described in Example 3, and the yield of rebaudioside M8 was calculated. The results showed that when the buffer solution was 100 mmol / L KPi pH 8.0 and 100 mmol / L NaCl, the yield of rebaudioside M8 could reach more than 32%. Figure 13 ).

[0082] Example 8 Effect of temperature on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase

[0083] The glycosylation coupling reaction system was placed at different temperatures (20~50℃) to carry out the reaction, and the effect of temperature on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined.

[0084] The cell lysate for the coupling reaction was prepared according to Example 6. The coupling reaction system consisted of 1 mL containing 40 mg / mL crude enzyme, 5 mM Reb D, 200 mM sucrose, 5% dimethyl sulfoxide (v / v), 100 mM KPi, and pH 8.0 (containing 100 mM NaCl). The reaction mixture was reacted at 20-50°C for 3 h; then the reaction was quenched by heating at 95°C for 5 min, and the reaction mixture was diluted with 4 times its volume of methanol; centrifuged at 20000×g for 5 min to remove precipitate; the supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis. The liquid chromatography detection method was performed as described in Example 3, and the yield of rebaudioside M8 was calculated. The results showed that when the temperature was 40-45°C, the yield of rebaudioside M8 could reach more than 43%. Figure 14 ).

[0085] Example 9: Effect of DMSO concentration on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase

[0086] 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.

[0087] The cell lysate for the coupling reaction was prepared according to Example 6. The coupling reaction system consisted of 1 mL containing 40 mg / mL crude enzyme, 5 mM Reb D, 200 mM sucrose, dimethyl sulfoxide (5%-25% (v / v)), 100 mM KPi, and pH 8.0 (containing 100 mM NaCl). The reaction mixture was reacted at 35°C for 3 h; then the reaction was quenched by heating at 95°C for 5 min, and the reaction mixture was diluted with 4 times its volume of methanol; centrifuged at 20000×g for 5 min to remove precipitate; the supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis. The liquid chromatography detection method was performed as described in Example 3, and the yield of rebaudioside M8 was calculated. The results showed that the yield of rebaudioside M8 remained stable when the DMSO concentration was between 5% and 15% (v / v). Figure 15 ).

[0088] Example 10 Effect of sucrose concentration on the coupling reaction of glycosyltransferase and sucrose synthase

[0089] Different concentrations of sucrose (50-800 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.

[0090] The cell lysate for the coupling reaction was prepared according to Example 6. The coupling reaction system consisted of 1 mL containing 40 mg / mL crude enzyme, 5 mM Reb D, 0-800 mM sucrose, 5% dimethyl sulfoxide, 100 mM KPi, and pH 8.0 (containing 100 mM NaCl). The reaction mixture was reacted at 35°C for 3 h; then the reaction was quenched by heating at 95°C for 5 min, and the reaction mixture was diluted with 4 times its volume of methanol; centrifuged at 20000×g for 5 min to remove precipitate; the supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis. The liquid chromatography detection method was performed as described in Example 3, and the yield of rebaudioside M8 was calculated. The results showed that when the sucrose concentration was 400 mmol / L, the yield of rebaudioside M8 could reach more than 34%. Figure 16 ).

[0091] Example 11 Effect of reaction time on the coupling reaction of glycosyltransferase and sucrose synthase

[0092] The effects of sucrose concentration on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase were determined by reacting the glycosylation coupling reaction system at different times (0-24 h).

[0093] Cell lysate for the coupling reaction was prepared according to Example 6. The coupling reaction system consisted of 20 mL containing 40 mg / mL crude enzyme, 5 mM Reb D, 400 mM sucrose, 5% dimethyl sulfoxide (v / v), 100 mM KPi, pH 8.0 (containing 100 mM NaCl), and was reacted at 45 °C for an optimized reaction time range of 0–24 h. Samples were taken at a series of time points (0–24 h, with 3 h intervals), and the reaction was quenched by heating at 95 °C for 5 min. The reaction mixture was then diluted with 4 times its volume of methanol. Centrifugation at 20,000 × g for 5 min was performed to remove precipitate. The supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis. The liquid chromatography method was performed as described in Example 3, and the yield of rebaudioside M8 was calculated. The results showed that, after 12 hours of reaction, at a concentration of 5 mmol / L rebaudioside D, 5.71 g / L of rebaudioside M8 was obtained with a yield of 88.52%. Figure 17 ).

[0094] Example 12 Sweetness test of rebaudioside M8

[0095] Rebaudioside D was used as a control in the sweetness test of rebaudioside M8. The rebaudioside D sample was purchased from Bidex Pharmaceuticals. The purity of rebaudioside M8 was 98%.

[0096] The SA402B electronic tongue system is used for sweetness analysis of glycosylated products. 20 mg of Reb D or 20 mg of the glycosylated product was thoroughly dissolved in 50 mL of ultrapure water (400 ppm) to prepare the sample solution for sweetness analysis. The SA402B electronic tongue system uses a lipid membrane sensor (GL1) and a reference electrode (Ag / AgCl). Before use, the sensor was soaked in the reference solution (30 mM KCl and 0.3 mM tartaric acid) for at least 24 h. Then, after equilibration in the reference solution, the activated sensor was sequentially immersed in the reference solution and the sample solution, and the membrane potential V was measured. r and V s The taste signal value (R) was calculated using the following formula. Each sample solution was measured four times, and the average of the last three measurements was taken as the test result:

[0097] (1)

[0098] As shown in Table 2, the analysis results indicate that the R (taste signal value) value of Reb M8 is higher than that of Reb D, meaning that the sweetness of Reb M8 is higher than that of Reb D, indicating that the single taste at position C-13 is superior. β -1,6-O-glycosylation can effectively improve the sweetness properties of Reb D, providing important information for the development of novel steviol glycoside natural sweeteners.

[0099] Table 2. Electronic tongue analysis of Reb M8 and Reb D

[0100] sample Reb D Reb M8 R (mV) -100 ± 1.56 -106.53 ± 1.76

[0101] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a steviol glycoside derivative, rebaudioside M8, characterized in that, The method includes (a) or (b): (a) Using UDP-glucose as a glycosyl donor and rebaudioside D as a substrate, the reaction is catalyzed by glycosyltransferase UGT94E13 and sucrose synthase; the NCBI accession number for the amino acid sequence of the glycosyltransferase UGT94E13 is MN944055.1; (b) Construct a recombinant strain expressing glycosyltransferase UGT94E13 and sucrose synthase, ferment and culture the recombinant strain, collect the bacterial culture, lyse and collect the supernatant, use UDP-glucose as glycosyl donor and rebaudioside D as substrate, and use the supernatant for catalytic reaction; The chemical structural formula of the steviol glycoside derivative rebaudioside M8 is shown below: 。 2. The method for preparing the steviol glycoside derivative rebaudioside M8 according to claim 1, characterized in that, The amino acid sequence of the sucrose synthase is an amino acid sequence of any origin that has sucrose synthase activity.

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