CN115433249A

CN115433249A - Preparation method of novel stevioside derivative rebaudioside L2

Info

Publication number: CN115433249A
Application number: CN202211014098.7A
Authority: CN
Inventors: 饶义剑; 平千; 郭保党; 张艳; 袁振波; 蒋杰娟; 杨丽烽
Original assignee: Jiangnan University
Current assignee: Guilin Layn Natural Ingredients Corp
Priority date: 2022-08-23
Filing date: 2022-08-23
Publication date: 2022-12-06

Abstract

The invention discloses a preparation method of a novel stevioside derivative rebaudioside L2, belonging to the field of biocatalytic synthesis. The invention obtains a glycosyltransferase YjiC with catalytic rebaudioside A monosaccharide acylation activity by mining, analyzes the complete structure of a product through LC-MS, 1D and 2D NMR spectra, and obtains a new product with the chemical structure of 13- [ (2-O-beta-D-glucopyranosyl-3-O-beta-D-glucopyranosyl-6-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl) oxy ] ent-kaur-16-en-19-oic acid beta-D-glucopyranosyl ester. And a glycosyltransferase YjiC and a sucrose synthase AtSuSy from Arabidopsis are constructed for coupling reaction, so that the rebaudioside L2 is efficiently catalytically synthesized by taking rebaudioside A as a substrate, and 30.94g/L of rebaudioside L2 is efficiently synthesized by taking 29.01g/L (30 mmol/L) of rebaudioside A as a substrate for reaction for 12h, wherein the yield of the rebaudioside L2 reaches 91.34%, and a new efficient and green way is provided for the production of the rebaudioside L2.

Description

Preparation method of novel stevioside derivative rebaudioside L2

Technical Field

The invention relates to a preparation method of a novel stevioside derivative rebaudioside L2, belonging to the technical field of biocatalytic synthesis.

Background

In recent years, the worldwide risk of caries, obesity, diabetes, hypertension, cardiovascular disease has increased, and therefore, consumer demand for low-calorie or non-calorie sweeteners has increased. Stevioside extracted from stevia rebaudiana is considered to be the most attractive sweetener at present due to its high sweetness (50-450 times that of sucrose), non-caloric and safety. In addition, rebaudioside has also been found to have important pharmacological activities, such as lowering blood sugar, lowering blood pressure, diuresis, anti-inflammatory, anti-tumor, and immunomodulating effects. Sixty or more steviol glycosides have been discovered from stevia rebaudiana, of which stevioside (5-10% of the dry weight of the leaves) and rebaudioside a (2-4% of the dry weight of the leaves) are the two most abundant components, and are also the main components of commercially available stevioside additives on the market today. Unfortunately, the consumption of steviol glycosides can be associated with an undeniable bitter taste, which also limits the commercial use of steviol glycosides to their success.

It has now been found that the amount and position of the sugar attached to the C-13 and/or C-19 position of the steviol glycoside can significantly affect sweetness and mouthfeel, but the specific structure relationship to sweetness has not been fully resolved. A more efficient way to solve this problem is to introduce a single glycosyl unit at different positions. Therefore, related researchers have performed many chemical and biological modifications on steviol glycosides to elucidate the relationship between structure and function of steviol glycosides and expect to obtain steviol glycosides with better sweetness quality. To date, various enzymes have been reported for glycosylation modification of steviol glycosides, such as cyclodextrin glycosyltransferase, glucanase, galactosidase, glucosidase, fructosidase, etc. However, these enzymes have the disadvantages of low yield, product mix, etc., and often introduce multiple glycosyl units on the substrate simultaneously, compared with UDP-glycosyltransferase with high conversion rate and regioselectivity. Therefore, the excavation of UDP-glycosyltransferase to realize the monosaccharide glycosylation of different positions of rebaudioside A has important significance for explaining the relation between the stevioside structure and the sweet taste quality.

Disclosure of Invention

In order to solve the problems, the invention discloses a glucoside transferase YjiC from bacillus for catalyzing rebaudioside A to synthesize a mono-glycosylation derivative rebaudioside L2, wherein the glucoside transferase YjiC can realize high-efficiency soluble expression in escherichia coli and has the activity of catalyzing rebaudioside A to synthesize rebaudioside L2 in the presence of uridine diphosphate glucose (UDPG). And through constructing a uridine diphosphate glucose UDPG circulating regeneration system, efficient biosynthesis of rebaudioside L2 is realized by utilizing an escherichia coli lysate, and an effective method is provided for industrial application of rebaudioside L2.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the first object of the invention is to provide a compound rebaudioside L2, wherein the chemical structural formula of the compound rebaudioside L2 is shown as follows:

it is a second object of the present invention to provide a recombinant bacterium which expresses a glycosyltransferase having an amino acid sequence having NCBI accession number WP _003232783.1.

In one embodiment, the recombinant bacterium further expresses a sucrose synthase.

In one embodiment, the amino acid sequence of the sucrose synthase may be an amino acid sequence having sucrose synthase activity from any source.

In one embodiment, the amino acid sequence of the sucrose synthase has NCBI accession No. NP _001031915.

In one embodiment, the recombinant bacterium uses escherichia coli as a host cell.

The third purpose of the invention is to provide a method for catalytically synthesizing rebaudioside L2, wherein the method uses UDP-glucose (UDPG) as a glycosyl donor to catalyze rebaudioside a to prepare and obtain rebaudioside L2; the composition is glycosyltransferase YjiC or the recombinant bacteria or one or more cell lysates of the recombinant bacteria.

In one embodiment, the method is to use rebaudioside A as a substrate and perform a catalytic reaction by using a cell lysate of the recombinant bacterium.

In one embodiment, the cell lysate is a supernatant obtained by cell lysis after the induction expression of the recombinant bacteria.

In one embodiment, the conditions of the catalytic reaction are: with 5-50mmol/L rebaudioside A, 50-800mmol/L sucrose, 5-25% (v/v) DMSO, 100mmol/L K ₂ HPO ₄ -KH ₂ PO ₄ Buffer solution, 100mmol/L NaCl as reaction system, and glycosylation reaction at 20-45 deg.C for 0-48h.

In one embodiment, the buffer has a pH of 5.5 to 9.0.

The invention also provides application of the glycosyltransferase YjiC or the recombinant bacterium or the method in preparation of a rebaudioside L2-containing product.

The fourth purpose of the invention is to provide a sweetener, wherein the sweetener contains the compound rebaudioside L2.

In one embodiment, the sweetener further comprises a flavoring agent.

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

The invention also provides application of the compound rebaudioside L2 or the sweetener in the fields of food, medicine or chemical industry.

Has the advantages that:

(1) The invention uses the nucleic acid sequence of the encoding glycosyltransferase YjiC to prepare the recombinant protein capable of catalyzing the glycosylation of rebaudioside A, and the prepared recombinant protein can synthesize the rebaudioside A mono-glycosylation derivative rebaudioside L2 by using UDPG as a glycosyl donor and rebaudioside A as a substrate. Provides a new analogue for clarifying the relation between the stevioside structure and the sweet taste.

(3) According to the invention, glycosyltransferase YjiC and sucrose synthase AtSuSy are combined to construct a UDPG circulating regeneration system. Through the optimization of the coupling reaction system conditions, the synthesis of 30.94g/L of rebaudioside L2 with high yield of 91.34% by using 29.01g/L (30 mmol/L) of rebaudioside A is realized.

(4) The constructed recombinant strain co-expresses glycosyltransferase derived from bacillus and sucrose synthase derived from arabidopsis thaliana, and the cell lysate prepared after the induction expression of the recombinant strain is utilized to catalyze rebaudioside A to synthesize rebaudioside L2, so that a glycosyl donor is not required to be added, and a cell penetrating agent is not required to be additionally used, so that the cost is remarkably reduced, and the method is green and environment-friendly.

Drawings

Fig. 1 shows the biosynthetic pathway for the production of rebaudioside L2 by the glycosyltransferase YjiC catalyzing rebaudioside a.

FIG. 2 shows the expression and purification analysis of the glycosyltransferase YjiC protein in example 2.

FIG. 3 is a UPLC analysis chart of the synthesis of rebaudioside L2 from rebaudioside A catalyzed by glycosyltransferase YjiC in example 3. Lane 1: marker; lane 2: no IPTG induced expression samples; lane 3: crude enzyme solution; lane 4: supernatant of the crude enzyme solution; lane 5: precipitating the crude enzyme solution; lane 6: purifying the transudate liquid; lane 7: washing the heteroprotein sample; lane 8: the protein of interest elutes the sample.

FIG. 4 is a rebaudioside L2 mass spectrometry analysis of the rebaudioside A glycosylation reaction product of example 3.

FIG. 5 is a NMR spectroscopic hydrogen spectrum of rebaudioside L2, a product of example 4.

FIG. 6 is a carbon spectrum of NMR spectroscopy analysis of rebaudioside L2, a product of example 4.

FIG. 7 is a magnetic resonance spectroscopy COSY spectrum of the product rebaudioside-L2 in example 4.

FIG. 8 is a nuclear magnetic resonance spectroscopy TOCSY spectrum of rebaudioside L2, the product of example 4.

FIG. 9 is an NMR spectroscopy HSQC spectrum of rebaudioside L2, the product of example 4.

FIG. 10 is a NMR spectroscopic HMBC spectrum of the product rebaudioside L2 of example 4.

FIG. 11 is a NMR spectroscopy ROESY spectrum of rebaudioside L2, a product of example 4.

FIG. 12 shows the protein expression analysis of lysates from YjiC-AtSuSy glycosylation coupling reaction in example 6. Lane 1: marker; lane 2: no IPTG induced expression samples; lane 3: crude enzyme solution; lane 4: supernatant of the crude enzyme solution; lane 5: and precipitating the crude enzyme solution.

FIG. 13 is a graph showing the effect of pH on buffer solutions for glycosylation coupling reactions in example 7.

FIG. 14 is a graph showing the effect of temperature on the glycosylation coupling reaction in example 8.

FIG. 15 is a graph showing the effect of DMSO concentration on the glycosylation coupling reaction in example 9.

FIG. 16 is a graph showing the effect of sucrose concentration on glycosylation coupling reaction in example 10.

FIG. 17 is a graph showing the effect of substrate concentration on glycosylation coupling reaction in example 11.

FIG. 18 shows the effect of reaction time on the glycosylation coupling reaction in example 12.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

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

The methods referred to in the following examples:

determination of the enzymatic properties of glycosyltransferases: kinetic analysis of rebaudioside A by glycosyltransferase YjiC was performed in a 200 μ L reaction system containing 5mM UDPG,10mM MnCl ₂ 50mM Bis-Tris pH 7.0 and 5 μ g purified protein sample, with rebaudioside A concentrations ranging from 0.5 to 8mM. The reaction temperature is 35 ℃, the reaction time is 20min, and the reaction is quenched by heating at 95 ℃ for 10min immediately after the reaction is finished. Diluting with 5 times volume of methanol, centrifuging at 20000 × g for 5min, and removing protein precipitate. The supernatant was filtered through a 0.22 μm filter and the sample was analyzed by UPLC.

Determination of rebaudioside L2 yield: preparing rebaudioside L2 standard solutions with different concentrations (1 mM, 2mM, 3mM, 4mM, 5mM and 6 mM), analyzing the standard solutions by using UPLC to obtain a rebaudioside L2 concentration standard curve equation of y =4450596.9x +346752.0 ² =0.99892. And (5) converting according to a standard curve to obtain the rebaudioside L2 yield. Yield = rebaudioside L2 actual yield/rebaudioside L2 theoretical yield.

Example 1 acquisition of glycosyltransferase YjiC Gene and construction of recombinant Strain

The amino acid sequence (accession number: WP _ 003232783.1) and the nucleic acid sequence (accession number: CP 053102.1) of Bacillus glycosyltransferase were downloaded from Genbank, and were genetically synthesized by Hakken Biotech Ltd and ligated to the polyclonal cleavage site of vector pET-21b (+), to obtain recombinant plasmid pET-21b (+) -YjiC.

The obtained plasmid pET-21b (+) -YjiC is subjected to sequencing identification and is transformed into a competent cell of Escherichia coli E.coli BL21 (DE 3), and an LB solid plate (10 g/L peptone, 5g/L yeast powder, 10g/L NaCl,20g/L agar powder) containing 100 mu g/mL ampicillin is adopted for screening, so that a recombinant strain E.coli BL2L (DE 3) pET-21b (+) -YjiC is obtained.

Example 2 inducible expression of recombinant Strain and purification of protein of interest

The recombinant strain E.coli BL2L (DE 3) pET-21b (+) -YjiC constructed in example 1 was inoculated into 1L 2 XYT liquid medium (16 g/L peptone, 10g/L yeast powder, 5g/L NaCl) containing 100. Mu.g/mL ampicillin and cultured at 135rpm at 37 ℃ to OD ₆₀₀ After the concentration is 0.6-0.8 ℃, the culture temperature is reduced to 18 ℃, isopropyl-beta-thiogalactoside (IPTG) with the final concentration of 0.1mmol/L is added, and the induction culture is carried out for 8 hours.

The induced expression was centrifuged (7000rpm, 7min,4 ℃ C.), the supernatant was discarded, and the cells were collected. The cells were resuspended in lysis buffer (50 mmol/L Tris-HCl pH 8.0, 300mmol/L NaCl,10mmol/L imidazole, 10% glycerol) per 10mL of lysis buffer per 1g of cells. Crushing by a high-pressure homogenizer, centrifuging the crushed bacterial liquid (40000 Xg, 30 min), and taking the supernatant to obtain a crude enzyme liquid.

Using the crude enzyme solution with Ni ⁺ And (3) carrying out affinity chromatography purification on the column, after the sample loading is finished, washing the hybrid protein by 10 times of volume of lysate, and eluting the target protein by using an elution buffer (50 mmol/L Tris-HCl pH 8.0, 300mmol/L NaCl,250mmol/L imidazole and 10% glycerol). The eluted target protein was collected and desalted by Desalting column (HistrpTM 5mL desaling) with Desalting buffer (25 mmol/L Tris-HCl,150mmol/L NaCl,10% glycerol). After desalting, the mixture was concentrated to 10mg/mL and subjected to the subsequent reaction. The purified protein was detected by 10% SDS-PAGE gel electrophoresis, and the results are shown in FIG. 2, and the pure enzyme with the correct size of the band-clear protein of interest was successfully obtained. K of YjiC to rebaudioside A is measured _m The value was 1.25. + -. 0.11mM _cat The value was 0.71. + -. 0.02s ^-1 。

Example 3 glycosylation reaction of YjiC to catalyze rebaudioside A reaction to synthesize rebaudioside L2

The purified glycosyltransferase YjiC obtained in example 2 was used in the glycosylation reaction (FIG. 1).

The glycosylation reaction was carried out in a 200. Mu.L reaction system as follows: 50mmol/L Tris-HCl pH 8.0,5mmol/L UDPG,10mmol/L MnCl ₂ 3mmol/L rebaudioside A, the concentration of pure enzyme YjiC obtained in example 2 was 10. Mu.M. The reaction was carried out at 35 ℃ for 20min. After the reaction, the reaction mixture was diluted with 5 volumes of methanol, centrifuged at 20000 Xg for 5min, filtered through a 0.22. Mu.M filter membrane, and the filtrate was subjected to ultra-high performance liquid chromatography (UPLC) detection and analysis. UPLC used waters BEH C18.7. Mu.M reverse column, 2. Mu.L sample size, 40 ℃ column temperature, mobile phase used A line: acetonitrile, line B: 1.38g/L NaH ₂ PO ₄ Buffer (pH 2.6), flow rate 0.3mL/min, specific procedure as shown in Table 1:

TABLE 1UPLC reaction procedure

As can be seen from the results of liquid phase analysis, as shown in FIG. 3, when compared with rebaudioside A standard, a significant new product was produced in the reaction system, and Mass Spectrometry (MS) analysis was performed on the reaction mixture (FIG. 4), and the negative ion mode results of LC-MS showed that there was a [ M-H ] at M/z 1127.4790] ^- Ion peak, corresponding to formula C ₅₀ H ₈₀ O ₂₈ It shows that the product is a monosaccharide derivative of rebaudioside A, with a [ M-Glc-H ] at M/z 965.4385] ^- Peak of ion. Since the ester bond at the C-19 position of steviol glycoside is more easily cleaved in ESI-MS to generate fragment ions, it is speculated that the new glucosyl unit is increased in the C-13-trisaccharide portion of rebaudioside A.

Example 4 structural identification of novel rebaudioside a monoglycosylated derivatives

The novel derivatives were prepared using glycosyltransferase YjiC for large scale (100 mL) glycosylation reactions under the same conditions as in example 3. Using semi-preparative high performance liquid chromatography systemsPurification was carried out using a Shim-pack GIST C18 column (10X 250mm,5 μm, SHIMADZU, japan) with a detection wavelength of 210nm, a mobile phase of a mixture of acetonitrile and water, a solvent flow rate of 5mL/min, a column temperature of 40 ℃ and an elution procedure of 0 to 35min of 26% acetonitrile isocratic elution. 85mg of high-purity product were obtained from the reaction mixture. Then, by 1D: ( ¹ H and ¹³ c) And 2D NMR (COSY, TCOSY, HSQC, HMBC, and ROESY) spectra were analyzed for the complete structure of the product (see FIGS. 5-11). Data were collected using a Bruker Avance III 600MHz spectrometer (Bruker BioSpin, karlsruhe, germany), ¹ the frequency of the H spectrum detection is 600MHz, ¹³ the C spectrum was 151MHz.1H-NMR has typical steviol glycoside signal characteristics. Peaks with chemical shifts less than 2.5ppm are derived from terpene aglycones and peaks with chemical shifts from 3-6ppm are derived from sugar rings.

From ¹ H and ¹ H- ¹³ the C HSQC spectrum shows delta _H 6.07(δ _C 95.56)、δ _H 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 Presence of 5 aberrant protons, confirming the presence of 5 saccharide units in the structure of the product. At the same time, at delta _H 6.07(J＝8.4Hz)、δ _H 5.40(J＝7.3Hz)、δ _H 5.25(J＝7.9Hz)、δ _H 4.98(J＝7.9Hz)、δ _H 4.90 The observation of a high J dipole moment at (J =6.8 Hz) indicates that the 5 glucose residues are all in the beta configuration. The chemical shifts of H and C of the new derivatives were assigned in detail by 1D and 2D HMR as shown in table 2. The new product is determined to be a glucosyl group connected with the C-13 position of the core of the rebaudioside A diterpene through a beta-1, 6-bond, and the new product is named rebaudioside L2. The structural formula is 13- [ (2-O-beta-D-glucopyranosyl-3-O-beta-D-glucopyranosyl-6-O-beta-D-glucopyranosyl) oxy)]ent-kaur-16-en-19-oic acidβ-D-glucopyranosyl ester。

TABLE 2 ¹ H and ¹³ c chemical shift Chart (pyridine-d) ₅ )

Example 5 construction of glycosyltransferase and sucrose synthase recombinant plasmids and recombinant strains

An amino acid sequence (accession number: NP-001031915) of a sucrose synthase AtSuSy derived from Arabidopsis thaliana and a nucleic acid sequence (accession number: NM-001036838.2) thereof were downloaded from Genbank, and codon optimization and gene synthesis were carried out with preference for Escherichia coli by York Biotechnology Ltd. The gene AtSuSy for coding sucrose synthase is connected to a polyclonal enzyme cutting site of pACYCDuet-1 to construct a recombinant plasmid pACYCDuet-1-AtSuSy, the obtained plasmid is sequenced and identified, and is co-transformed into a competent cell of Escherichia coli E.coli BL21 (DE 3) with pET-21b (+) -YjiC, an LB solid plate (10 g/L peptone, 5g/L yeast powder, 10g/L NaCl,20g/L agar powder) containing 100 mu g/mL ampicillin and 34 mu g/mL chloramphenicol is adopted for screening, and a recombinant strain E.coli BL2L (DE 3) YjiC-AtSuSy is obtained.

EXAMPLE 6 preparation of cell lysate from glycosyltransferase and sucrose synthase coupling reaction

The recombinant strain E.coli BL2l (DE 3) YjiC-AtSuSy constructed in example 5 was inoculated into 5L 2 XYT medium containing 100. Mu.g/mL ampicillin and 34. Mu.g/mL chloramphenicol, and cultured with shaking at 37 ℃ and 135 rpm. After the thallus grows to OD ₆₀₀ Cooling to 18 deg.C when the value is 0.6-0.8, adding isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 0.2mM, inducing expression at 18 deg.C for 8 hr, centrifuging at 7000 Xg for 7min, collecting thallus, and treating with lysis buffer (100 mM K) ₂ HPO ₄ -KH ₂ PO ₄ (KPi) pH 8.0,100mM NaCl) three times and then resuspended. Cells were disrupted with a high pressure homogenizer. Immediately, the cells were centrifuged at 40000 Xg for 30min to remove cell debris. And taking the supernatant to obtain the cell lysate of the coupling reaction. And performing protein gel detection.

The results are shown in FIG. 12, which shows that both glycosyltransferase and sucrose synthase are well expressed. The protein concentration in the cell lysate was determined using a Nano-Drop 2000UV-Vis spectrophotometer. The prepared cell lysate is subpackaged and stored at-80 ℃, or is directly used for coupling reaction.

Example 7 Effect of pH on glycosyltransferase and sucrose synthase glycosylation coupling reactions

Placing the glycosylation coupling reaction system in buffer solutions with different pH values for reaction, and determining the influence of the pH on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase, wherein the selected buffer solution is 100mmol/LBis-Tris pH 5.5-7.5 and 100mmol/L NaCl;100mmol/L KPi pH 5.5-8.0, 100mmol/L NaCl and 100mmol/L Tris-HCl pH 7.5-9.0, 100mmol/L NaCl.

Preparing a coupling reaction cell lysate as described in example 6, the glycosylation coupling reaction system was 1mL containing 40mg/mL of the coupling reaction cell lysate, 30mmol/L of rebaudioside A, 200mmol/L of sucrose and 5% DMSO (v/v), buffer; reacting for 6 hours at 35 ℃. After the reaction is finished, 5 times of methanol is added to stop the reaction, then methanol is added to dilute the reaction by 6 times, 20000 Xg is centrifuged for 5min, a 0.22 mu M filter membrane is filtered, and then UPLC is used for detection and analysis. The liquid phase assay was performed as described in example 3 and the rebaudioside L2 yield was calculated, showing that the rebaudioside L2 yield can reach 55% or more when the buffer was 100mmol/L KPi pH 7.5 and 100mmol/L NaCl (fig. 13).

EXAMPLE 8 Effect of temperature on glycosyltransferase and sucrose synthase glycosylation coupling reactions

The glycosylation coupling reaction system is placed in different temperatures (20-45 ℃) for reaction, and the influence of the temperature on the glycosylation coupling reaction of the glycosyl transferase and the sucrose synthase is measured.

Preparation of a coupling reaction cell lysate as described in example 6, the coupling reaction system was 1mL containing 40mg/mL of the coupling reaction cell lysate, 30mmol/L of rebaudioside A, 200mmol/L of sucrose, 10% DMSO (v/v) and 100mmol/L of KPi pH 7.5, 100mmol/L of NaCl, and reacted for 6h. After the reaction is finished, 5 times of methanol is added to stop the reaction, then methanol is added to dilute the reaction by 6 times, 20000 Xg is centrifuged for 5min, a 0.22 mu M filter membrane is used for suction filtration, and UPLC is used for detection and analysis. The liquid phase assay was performed as described in example 3, and the rebaudioside L2 yield was calculated, showing that the rebaudioside L2 yield can reach 50% or more when the temperature was 30-40 ℃ (fig. 14).

Example 9 Effect of DMSO concentration on glycosyltransferase and sucrose synthase glycosylation coupling reactions

Adding DMSO (5-25% (v/v)) with different concentrations into a glycosylation coupling reaction system for reaction, and determining the influence of the DMSO concentration on glycosylation coupling reaction of glycosyltransferase and sucrose synthase.

A coupling reaction cell lysate is prepared as described in example 6, and the coupling reaction system is 1mL, which contains 40mg/mL of coupling reaction cell lysate, 30mmol/L of rebaudioside A, 200mmol/L of sucrose, DMSO (5% -25% (v/v)), and 100mmol/L of KPi pH 7.5, 100mmol/L of NaCl, and reacts for 6h at 35 ℃. After the reaction is finished, 5 times of methanol is added to stop the reaction, then the reaction is diluted by 6 times, 20000 Xg is centrifuged for 5min, a filter membrane of 0.22 mu M is used for suction filtration, and UPLC is used for detection and analysis. The liquid phase assay was performed as described in example 3 and the rebaudioside L2 yield was calculated, showing that the rebaudioside L2 yield can reach more than 55% when the DMSO concentration was between 5% and 10% (v/v) (fig. 15).

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

Adding sucrose (50-800 mmol/L) with different concentrations into a glycosylation coupling reaction system for reaction, and determining the influence of the sucrose concentration on glycosylation coupling reaction of glycosyl transferase and sucrose synthase.

A coupled reaction cell lysate is prepared as described in example 6, wherein the coupled reaction system is 1mL, and the coupled reaction cell lysate contains 40mg/mL of coupled reaction cell lysate, 30mmol/L of rebaudioside A, sucrose (50-800 mmol/L), 10% (v/v) DMSO and 100mmol/L KPi, pH 7.5, 100mmol/L NaCl, and the reaction is carried out at 35 ℃ for 6h. After the reaction is finished, 5 times of methanol is added to stop the reaction, then the reaction is diluted by 5 times, 20000 Xg is centrifuged for 5min, a 0.22 mu M filter membrane is used for suction filtration, and detection and analysis are carried out by UPLC. The liquid phase assay was performed as described in example 3, and the rebaudioside L2 yield was calculated, showing that the rebaudioside L2 yield can reach more than 60% when the sucrose concentration was 250-600 mmol/L (fig. 16).

Example 11 Effect of rebaudioside A concentration on glycosyltransferase and sucrose synthase coupling reaction substrates

Adding rebaudioside A (5-50 mmol/L) with different concentrations into a glycosylation coupling reaction system for reaction, and determining the influence of sucrose concentration on glycosylation coupling reaction of glycosyl transferase and sucrose synthase.

A coupled reaction cell lysate is prepared as described in example 6, and the coupled reaction system is 1mL, which contains 40mg/mL coupled reaction cell lysate, rebaudioside A (5-50 mmol/L), 500mmol/L sucrose, 10% (v/v) DMSO, and 100mmol/L KPi pH 7.5, 100mmol/L NaCl, and reacts for 6h at 35 ℃. After the reaction is finished, 5 times of methanol is added to stop the reaction, then the reaction is diluted by 5 times, 20000 Xg is centrifuged for 5min, a 0.22 mu M filter membrane is used for suction filtration, and detection and analysis are carried out by UPLC. The liquid phase detection method was performed as described in example 3, and the yield of rebaudioside L2 was calculated, and the results showed that 24.00g/L of rebaudioside L2 was obtained at a yield of 70.86% at a substrate concentration of 30mmol/L (fig. 17).

EXAMPLE 12 Effect of reaction time on glycosyltransferase and sucrose synthase coupling reactions

The glycosylation coupling reaction system is reacted for different time (0-24 h), and the influence of the sucrose concentration on the glycosylation coupling reaction of the glycosyltransferase and the sucrose synthase is measured.

The coupling reaction cell lysate is prepared as described in example 6, the coupling reaction system is 20mL, and the coupling reaction cell lysate contains 40mg/mL coupling reaction cell lysate, 30mmol/L rebaudioside A, 400mmol/L sucrose, 10% (v/v) DMSO, 100mmol/L KPi pH 7.5 and 100mmol/L NaCl, and the reaction is carried out at 35 ℃, and the optimized range of the reaction time is 0-24h. After the reaction is finished, 5 times of methanol is added to stop the reaction, then the reaction is diluted by 5 times, 20000 Xg is centrifuged for 5min, and the filtration membrane with the volume of 0.22 mu M is filtered and then the detection and analysis are carried out by Ultra Performance Liquid Chromatography (UPLC). The liquid phase assay method was performed as described in example 3 and the rebaudioside L2 yield was calculated and the results showed that finally at a reaction time of 12h, 30.94g/L rebaudioside L2 was obtained at a yield of 91.34% at a rebaudioside a concentration of 30mmol/L (fig. 18).

Example 13 sweetness testing of rebaudioside L2

Sweetness testing of rebaudioside L2 was performed using sucrose as a control. Sucrose samples were purchased from pharmaceutical group, ltd, china. Wherein the purity of the rebaudioside L2 is 95 percent.

Three different concentrations of aqueous sucrose solutions were prepared at room temperature as controls: 1%, 5%, 10% (w/v). And 150 and 300ppm rebaudioside L2 aqueous solutions were prepared. 10mL of each sample solution was sampled in a 30mL disposable drinking cup, and 9 trained and experienced volunteers were subjected to sweetness evaluation (blind evaluation), and the evaluation results were averaged from the scores of the volunteers. In the evaluation, the sweetness is measured by 10 points (the sweetness is 10 points as 10% of sucrose, 5 points as 5% of sucrose, and so on) with 10 mass percent of sucrose aqueous solution, and the sweetness is 0 point which can not be detected completely.

Blind scoring results show that the sweetness scores for rebaudioside L2 at two different concentrations (150 and 300 ppm) are consistent, being 4.5 and 9.2, respectively. The results indicate that rebaudioside L2 is about 300 times more sweet than sucrose.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A compound rebaudioside L2, wherein the compound rebaudioside L2 has a chemical structure as shown in the following formula:

2. a recombinant bacterium which expresses glycosyltransferase YjiC; the glycosyltransferase YjiC has the amino acid sequence with NCBI accession number WP _003232783.1.

3. The recombinant bacterium of claim 2, wherein the recombinant bacterium further expresses a sucrose synthase.

4. The recombinant bacterium according to claim 3, wherein the amino acid sequence of sucrose synthase is an amino acid sequence having sucrose synthase activity from any source.

5. The recombinant bacterium according to any one of claims 2 to 4, wherein Escherichia coli is used as a host cell.

6. The method for catalytically synthesizing rebaudioside L2 is characterized in that UDP-glucose is used as a glycosyl donor, and the rebaudioside A is catalyzed into rebaudioside L2 by using the composition; the composition is one or more of glycosyltransferase YjiC, the recombinant bacterium according to any one of claims 2 to 4, or a cell lysate of the recombinant bacterium according to any one of claims 2 to 4.

7. The method of claim 6, wherein the cell lysate is a supernatant obtained by cell lysis after the recombinant bacterium is induced to express.

8. A sweetener comprising the compound rebaudioside L2 according to claim 1.

9. The compound of claim 1, rebaudioside L2, or the sweetener of claim 8, for use in the food, pharmaceutical, or chemical arts.

10. Use of a glycosyltransferase YjiC or a recombinant bacterium according to any one of claims 2 to 4 or a method according to claim 6 or 7 for the preparation of a rebaudioside L2-containing product; the glycosyltransferase YjiC has the amino acid sequence with NCBI accession number WP _003232783.1.