Method for efficiently preparing rebaudioside m by using bio-enzymatic method

Abstract

Disclosed is a method for efficiently preparing rebaudioside M by using a bio-enzymatic method. The preparation method comprises: adding rebaudioside A, uridine diphosphate glucose, glycosyltransferase, and a salt into a buffer solution, mixing to obtain a mixed solution, performing an enzyme-catalyzed reaction while stirring, and obtaining said rebaudioside M. The amino acid sequence of the glycosyltransferase is one or more of those shown in SEQ ID NO. 1-12. The glycosyltransferase can also be coupled with sucrose synthase for catalysis, so as to use sucrose as a glycosyl donor to, in one step, catalyze rebaudioside A to generate rebaudioside M.

Description

Method for efficiently preparing rebaudioside M using bioenzymatic method Technical Field

[0001] The present invention belongs to the technical field of biocatalytic conversion, and in particular relates to a method for efficiently preparing rebaudioside M by utilizing a bioenzymatic method. Background Art

[0002] A class of green, healthy natural sweeteners, represented by steviol glycosides, has been found to be widely used as a sugar substitute. Steviol glycosides are natural sweeteners extracted from the leaves of the Stevia rebaudiana plant through a plant-based method. Stevia is native to the pristine forests of Paraguay and Brazil in South America. For centuries, locals have ground the leaves and used them as a sweetener. This sweetness is primarily derived from the mixture of steviol glycosides contained in the leaves. Steviol glycosides are a glycoside mixture composed of several diterpenoid glycoside compounds. Their chemical structure consists of a sugar ligand linked to a steviol aglycone. The glycosidic bonds in steviol glycosides are largely indigestible, releasing very little energy, making them very attractive sugar substitutes. Stevioside and rebaudioside A are the most abundant compounds in Stevia leaves. Stevioside and rebaudioside A are 200 times sweeter than sucrose and were among the first sweeteners to enter commercial use. However, stevioside and rebaudioside A bind to the bitter taste receptors hTAS2R4 and hTAS2R14 in taste buds, resulting in a pronounced bitter aftertaste and an unpleasant taste. This has reduced their acceptance, impacting their application and market acceptance. Rebaudioside M, present in very small amounts (0.3%) in stevia leaves, offers a rapid, pure sweetness with minimal calories. Its sensory properties are most similar to sucrose, making it of great research interest in the food and pharmaceutical industries. It is expected to replace sucrose, a traditional sugar source, as a widely used natural sweetener, earning it the title of "next-generation sweetener."

[0003] The currently commercialized product, rebaudioside A, is inferior in sweetness and flavor to rebaudioside M. However, rebaudioside M, the most flavorful of the stevia leaves, is severely lacking in stevia leaves, with a very low natural content. Unlike the more abundant steviosides and rebaudioside A, it cannot be commercially extracted and purified directly from stevia leaves. This limits the large-scale production and supply of rebaudioside M. Rebaudioside M production can be increased through genetically modified stevia or microbial methods, but both methods require a thorough understanding of the biosynthetic pathway of rebaudioside M, particularly the glycosylation process.

[0004] The biosynthetic pathway of steviol glycosides is now largely elucidated, and a series of glycosyltransferases (UGTs) have been identified that utilize uridine diphosphate glucose (UDP-glucose, UDPG) as a glycosyl donor. Analysis of the rebaudioside M biosynthetic pathway revealed that glycosylation at the C19-carboxyl position of rebaudioside A, forming a β(1-2) glycosidic bond at this position, is essential for the production of rebaudioside M and determines its natural abundance. Glycosylation of steviol glycosides relies on four glycosyltransferases (UGTs) located in the cytoplasm of Stevia rebaudiana. These UGTs are annotated as members of the GT1 family in the Carbohydrate-Active Enzymes (CAZy) database (www.cazy.org) and utilize uridine diphosphate glucose (UDP-glucose) as a glycosyl donor. Glycosyltransferases UGT85C2 and UGT74G1 attach the first glucosyl group to the C13 hydroxyl group and C19 carboxyl group of the steviol aglycone via a β-1 glycosidic bond, respectively. UGT91D2 then adds a glycosyl group to the C2 position of the first glucosyl group via a β(1-2) glycosidic bond, and finally, UGT76G1 adds a glycosyl group to the C3 position of the first glucosyl group. Zhang et al. found that stevia's inherent lack of the ability to form β(1-2) glycosidic bonds at the C19-carboxyl group of steviol glycosides is the direct cause of the low natural content of steviol glycoside DM.

[0005] Currently, the production of rebaudioside M through glycosylation reaction using rebaudioside A as a substrate usually requires a two-step conversion. First, rebaudioside A is glycosylated to produce rebaudioside D, and then further glycosylated to produce rebaudioside M. The disadvantage of the two-step catalytic method for producing rebaudioside M is that it easily causes the accumulation of the intermediate product rebaudioside D, which is not conducive to subsequent separation and purification. In addition, the two-step method for catalyzing rebaudioside A to produce rebaudioside M usually requires multiple enzymes, and the steps are relatively cumbersome.

[0006] Typically, glycosylation reactions catalyzed by UDP-glycosyl donor-dependent glycosyltransferases (UGTs) require the use of expensive and unstable UDP-glycosyl donors, such as UDP-glucose (UDPG). UDPG is too expensive, and its use as a glycosyl donor incurs huge costs, making it unsuitable for large-scale industrial production. This severely limits the industrial application of UGTs.

[0007] Patent CN110734944B applied for by Tianjin University - a one-step method for synthesizing rebaudioside M. This invention uses rebaudioside A as a substrate, adds uridine diphosphate glucose, magnesium sulfate or magnesium chloride, and methanol, and utilizes recombinant engineered bacteria UGT1 and UGT2 that can secrete and express glycosyltransferases to catalyze the reaction to obtain rebaudioside M. This method adds a large amount of metal ions and organic solvents, which is not conducive to subsequent separation and purification and food-grade production. In addition, expensive uridine diphosphate glucose is added to the reaction, and the production cost is too high. Patent CN109750071A, filed by Nanjing University of Technology, describes a method for biocatalytically synthesizing rebaudioside M. This method utilizes tomato-derived UDP-glycosyltransferase and potato-derived sucrose synthase to glycosylate stevioside to synthesize rebaudioside E. Subsequently, rebaudioside E is further glycosylated to synthesize rebaudioside M using stevia-derived UDP-glycosyltransferase and potato-derived sucrose synthase. This method requires the generation of rebaudioside E before further generating rebaudioside M. This method requires two or more catalytic reactions to obtain the final product, resulting in slow reaction rates, low catalytic efficiency, and a high number of byproducts, making separation and purification difficult.

[0008] Therefore, the current existing technology for producing rebaudioside M has the following shortcomings:

[0009] 1) The plant extraction method has complicated steps, low yield, high cost, and difficulty in separation and purification.

[0010] 2) The plant extraction method requires a large amount of stevia, which wastes arable land.

[0011] 3) Chemical synthesis produces a large amount of harmful gases and waste liquids, which is not conducive to environmental protection.

[0012] 4) The catalytic reaction system contains a large amount of metal ions and organic solvents, which is not conducive to subsequent separation and purification and food-grade production.

[0013] 5) Two or more catalytic reactions are required to obtain the final product, which has a slow reaction rate, low catalytic efficiency, and a large number of by-products, making it difficult to separate and purify.

[0014] 6) The sugar donor usually required is expensive uridine diphosphate glucose, which has high production costs and is not suitable for large-scale industrial production.

[0015] 7) It requires the addition of multiple enzyme preparations for catalytic reaction, resulting in poor specificity and low yield.

[0016] Therefore, there is an urgent need for a method for producing rebaudioside M with high efficiency and low cost.

[0017] Summary of the Invention

[0018] In order to overcome the deficiencies of the prior art, the present invention aims to provide a method for efficiently preparing rebaudioside M using a bioenzymatic method.

[0019] The method provided by the present invention uses gene mining to obtain a glycosyltransferase, then constructs an engineered bacterium for protein expression. The enzyme then catalyzes the in vitro glycosylation reaction using rebaudioside A as a substrate to synthesize rebaudioside M in a single step. This synthesis method has high catalytic efficiency, short reaction time, a single, easily separable product, and great potential for industrial application. Furthermore, the glycosyltransferase provided by the present invention can also be coupled with sucrose synthase for catalytic conversion of rebaudioside A to rebaudioside M in a single step using sucrose as the glycosyl donor.

[0020] The method provided herein for efficiently preparing rebaudioside M using a bioenzymatic method utilizes a glycosyltransferase with high catalytic efficiency to catalyze the production of rebaudioside M in a single step using rebaudioside A as a substrate, achieving efficient biosynthesis of rebaudioside M. This method avoids the production of the intermediate product, rebaudioside D, facilitates subsequent separation and purification, and streamlines the catalytic reaction process. This method utilizes a bioenzymatic method to efficiently synthesize rebaudioside M, a high-sweetness, low-calorie natural sweetener.

[0021] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0022] The present invention provides a method for efficiently preparing rebaudioside M using a bioenzymatic method, comprising the following steps:

[0023] Rebaudioside A, uridine diphosphate glucose, glycosyltransferase, and salt are added to a buffer solution and mixed evenly to obtain a mixed solution, and an enzyme-catalyzed reaction is carried out under stirring to obtain the rebaudioside M.

[0024] Furthermore, in the mixed solution, the concentration of rebaudioside A is 1-100 g / L;

[0025] Preferably, in the mixed solution, the concentration of rebaudioside A is 1 g / L.

[0026] Furthermore, in the mixed solution, the concentration of uridine diphosphate glucose is 0.5-20 mM;

[0027] Preferably, in the mixed solution, the concentration of UDP-glucose is 1 mM.

[0028] Furthermore, the salt is one or more of calcium salt, cobalt salt, iron salt, magnesium salt, manganese salt, ammonium salt, nickel salt, and zinc salt;

[0029] Preferably, the salt is one or more of calcium salt, magnesium salt, manganese salt and iron salt;

[0030] More preferably, the salt is a magnesium salt.

[0031] Furthermore, in the mixed solution, the concentration of salt is 0.1-10 mM;

[0032] Preferably, in the mixed solution, the concentration of salt is 1 mM.

[0033] Further, the pH of the buffer solution is 5.0-9.0;

[0034] Preferably, the pH of the buffer is 6.0-8.0;

[0035] Further preferably, the pH of the buffer solution is 8.0;

[0036] Furthermore, the stirring rate in the stirring state is 50-220 rpm;

[0037] Preferably, the stirring rate in the stirring state is 200 rpm.

[0038] Furthermore, the temperature of the enzyme-catalyzed reaction is 20-45° C., and the time of the enzyme-catalyzed reaction is 1-5 h.

[0039] Preferably, the temperature of the enzyme-catalyzed reaction is 30-37°C.

[0040] Further preferably, the temperature of the enzyme catalyzed reaction is 37°C.

[0041] Furthermore, in the mixed solution, the concentration of the glycosyltransferase is 1-20 g / L;

[0042] Preferably, in the mixed solution, the concentration of the glycosyltransferase is 5 g / L.

[0043] Furthermore, the sequence of the glycosyltransferase is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.

[0044] Preferably, the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO.1.

[0045] In the method provided by the present invention, the glycosyltransferase used has a high efficiency in catalyzing the glycosylation reaction. With uridine diphosphate glucose (UDPG) as the glycosyl donor, the enzyme can well catalyze the glycosylation of rebaudioside A to produce the high-value sweetener rebaudioside M in one step without the need to add any other enzymes.

[0046] In the method provided by the present invention, the glycosyltransferase used has an optimum pH of 8.0 and an optimum reaction temperature of 37°C for the glycosylation reaction. It is a metal ion-dependent enzyme, Mg 2+ , Ca 2+ 、Mn 2+ The catalytic efficiency of the enzyme is significantly promoted, among which Mg 2+ The promoting effect is the strongest.

[0047] In the method provided by the present invention, the optimal reaction conditions of the enzyme are: the final concentrations of various substances in the reaction system are 1 g / L rebaudioside A, 1 mM UDPG, 5 mM MgCl2, the reaction temperature is 37°C, the rotation speed is 220 rpm, and the reaction time is 1-2 hours. Under the optimal reaction conditions, the enzyme has a high catalytic efficiency in catalyzing the glycosylation of rebaudioside A to produce rebaudioside M, and the conversion rate can reach more than 95%.

[0048] The present invention provides another method for efficiently preparing rebaudioside M using a bioenzymatic method, comprising the following steps:

[0049] Rebaudioside A, sucrose, UDP (5-uridine diphosphate disodium salt), glycosyltransferase, sucrose synthase, and salt are added to a buffer solution and mixed evenly to obtain a mixed solution, and an enzyme-catalyzed reaction is carried out under stirring to obtain the rebaudioside M.

[0050] Furthermore, in the mixed solution, the concentration of rebaudioside A is 1-100 g / L;

[0051] Preferably, in the mixed solution, the concentration of rebaudioside A is 10 g / L.

[0052] Furthermore, in the mixed solution, the concentration of sucrose is 1-500 mM;

[0053] Preferably, in the mixed solution, the concentration of sucrose is 100 mM.

[0054] Furthermore, in the mixed solution, the concentration of UDP (5-uridine diphosphate disodium salt) is 0.1-20 mM;

[0055] Preferably, in the mixed solution, the concentration of UDP (5-uridine diphosphate disodium salt) is 2 mM.

[0056] Furthermore, the salt is one or more of calcium salt, cobalt salt, iron salt, magnesium salt, manganese salt, ammonium salt, nickel salt, and zinc salt;

[0057] Preferably, the salt is one or more of calcium salt, magnesium salt, manganese salt and iron salt;

[0058] Furthermore, in the mixed solution, the concentration of salt is 0.1-10 mM;

[0059] Preferably, in the mixed solution, the concentration of salt is 0.1-5 mM.

[0060] More preferably, in the mixed solution, the salt concentration is 1 mM.

[0061] Further, the pH of the buffer solution is 4.0-9.0;

[0062] Preferably, the pH of the buffer is 6.0-8.0;

[0063] Further preferably, the pH of the buffer solution is 8.0;

[0064] Furthermore, the stirring rate in the stirring state is 50-220 rpm;

[0065] Preferably, the stirring rate in the stirring state is 200 rpm.

[0066] Furthermore, the temperature of the enzyme-catalyzed reaction is 20-45° C., and the time of the enzyme-catalyzed reaction is 1-5 h.

[0067] Preferably, the temperature of the enzyme-catalyzed reaction is 30-37°C.

[0068] Further preferably, the temperature of the enzyme catalyzed reaction is 37°C.

[0069] Furthermore, in the mixed solution, the concentration of the glycosyltransferase is 1-20 g / L;

[0070] Preferably, in the mixed solution, the concentration of the glycosyltransferase is 5 g / L.

[0071] Furthermore, the sequence of the glycosyltransferase is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.

[0072] Preferably, the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO.1.

[0073] Furthermore, in the mixed solution, the concentration of the sucrose synthase is 1-10 g / L;

[0074] Preferably, in the mixed solution, the concentration of the sucrose synthase is 2 g / L.

[0075] Furthermore, the amino acid sequence of the sucrose synthase is one or more of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20.

[0076] Preferably, the amino acid sequence of the sucrose synthase is shown as SEQ ID NO.13.

[0077] The second method provided by the present invention for efficiently preparing rebaudioside M using a bioenzymatic method uses sucrose as a glycosyl donor. In the presence of UDP, the glycosyltransferase can be coupled with sucrose synthase for catalytic conversion, thereby converting rebaudioside A into a high-value sweetener, rebaudioside M, in one step without generating other intermediates or by-products. In this method, the optimal pH for the dual-enzyme coupled catalytic reaction is 8.0, the optimal reaction temperature is 37°C, and the Mg content is 0.1%. 2+ The catalytic efficiency of the coupled catalytic reaction is significantly improved. In this method, the reaction time of the dual enzyme coupled catalysis is short, and rebaudioside A is completely converted into rebaudioside M within 2 hours.

[0078] This method utilizes the coupled catalytic action of glycosyltransferase and sucrose synthase, uses sucrose as the glycosyl donor to replace expensive UDPG, and efficiently catalyzes RA to generate RM in one step, thereby reducing production costs.

[0079] Both methods provided by the present invention utilize glycosyltransferases with high catalytic efficiency, and can catalyze the production of rebaudioside M in one step using rebaudioside A as a substrate, thereby achieving efficient biosynthesis of rebaudioside M. These two methods can reduce impurities such as metal ions and byproducts in the system, lower the cost of separation and purification, and thus achieve green biosynthesis of rebaudioside M.

[0080] In the second method provided by the present invention, cheap sucrose is used as the glycosyl donor, which greatly reduces the production cost.

[0081] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0082] (1) In the method provided by the embodiment of the present invention, the enzyme used can efficiently catalyze rebaudioside A to produce rebaudioside M in one step under optimal reaction conditions. No other enzymes need to be added during the catalytic process. The enzyme can specifically catalyze the glycosylation reaction to produce rebaudioside M without intermediates or other by-products. The reaction time is short and the catalytic efficiency is high. The yield of the product rebaudioside M can reach more than 95%, which is conducive to the separation and purification of the product.

[0083] (2) In the method provided by the embodiment of the present invention, rebaudioside A can be catalyzed to rebaudioside M in one step under optimal reaction conditions through dual enzyme coupling catalysis, without the generation of by-products, with good specificity, which is conducive to the subsequent separation and purification of the product. Sucrose is used as the glycosyl donor, the production cost is low, and it is conducive to the large-scale biosynthesis of rebaudioside M.

[0084] (3) In the method provided in the embodiment of the present invention, the final reaction system contains only trace metal ions (Mg 2+ ), no intermediates or by-products, extremely low impurity content and easy separation, which is conducive to subsequent separation and purification in industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] FIG1 is a graph showing the catalytic efficiency of glycosyltransferases at different pH values;

[0086] FIG2 is a graph showing the catalytic efficiency of glycosyltransferases at different temperatures;

[0087] FIG3 is a graph showing the effects of different metal ions on the catalytic efficiency of glycosyltransferases;

[0088] FIG4 is a liquid phase detection diagram of the synthesis of rebaudioside M from rebaudioside A catalyzed by using UDPG as a glycosyl donor;

[0089] Figure 5 is a liquid phase detection diagram of the synthesis of rebaudioside M from rebaudioside A catalyzed by sucrose as the glycosyl donor. DETAILED DESCRIPTION

[0090] The following examples further illustrate the specific implementation of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes not specifically described below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0091] Example 1: Gene mining

[0092] First, glycosyltransferase (UGTs) genes were used as probe sequences, and several homologous sequences were obtained through homology search. Multiple sequence alignment was performed using MEGA software (MEGA-X). Then, an evolutionary tree was constructed using the sequences after extracting conserved sites. The evolutionary tree can intuitively display the evolutionary relationship of each homologous sequence. Several corresponding enzyme protein gene sequences that were close to the probe sequence species were selected as potential candidate genes. Then, molecular substrate docking was performed after Rosetta homology modeling, and free energy, binding energy, etc. were calculated to further screen the candidate gene sequences for subsequent functional verification.

[0093] Example 2: Recombinant plasmid construction

[0094] The glycosyltransferase gene selected above was sequence and codon optimized, and the glycosyltransferase gene fragment selected above was synthesized (six his tags were added to the C-terminus of the gene fragment during the synthesis process to facilitate the subsequent use of Ni 2+ The recombinant enzyme was isolated and purified by affinity chromatography), and the recombinant gene fragment was then ligated into the E. coli expression vector pET28a using restriction endonucleases BamH I and EcoR I to obtain a recombinant plasmid carrying the target gene.

[0095] The heat shock transformation method was used to construct recombinant E. coli. Several synthesized recombinant plasmids were transformed into E. coli DH5α (for PCR amplification of the recombinant plasmid) and E. coli BL21 (DE3) (for heterologous expression of the recombinase protein) competent cells to obtain the corresponding recombinant E. coli containing the target gene.

[0096] Example 3: Expression of recombinase

[0097] (1) Activation culture: 5 μL of glycerol bacteria was inoculated into 5 mL of LB liquid culture medium containing Kan resistance (final concentration of 100 μg / mL), and cultured overnight in a biochemical incubator at a temperature of 37°C and a rotation speed of 220 rpm (culture time is 16 hours).

[0098] (2) Scale-up culture: Use a pipette to transfer 8 mL of activated bacterial liquid into a 1 L conical flask containing 800 mL of 2×YT liquid culture medium (containing Kan resistance). Culture the culture in a biochemical incubator at a temperature of 37°C and a rotation speed of 220 r / min for a certain period of time, and monitor the OD value at the same time.

[0099] (3) Induction of expression: When the OD value of the bacterial solution reaches 0.6-0.8, stop the culture at 37°C, add 400 μL of IPTG (0.5 mM) inducer to the conical flask, change the temperature to 16°C, keep the rotation speed unchanged, and continue to culture at low temperature in a constant temperature shaking incubator for 16-20 hours to induce overexpression of the recombinase.

[0100] (4) Centrifugation: The recombinant enzyme bacterial solution after low-temperature induction expression in step (3) was centrifuged in a floor-standing centrifuge at 4°C, 6000 rpm, and 10 min. After centrifugation, the supernatant was discarded, leaving the bacterial pellet. The cell pellet was resuspended and washed with 50 mM PBS buffer, pH 8.0, to 40 ml, and centrifuged at 4°C, 8000 rpm for 10 min. The supernatant was discarded, and the bacterial pellet was collected.

[0101] (5) Bacterial disruption: Add 50 mL of 50 mM PBS buffer (pH 8.0) to the washed bacterial pellet and resuspend it. Then, disrupt the cells using a high-pressure, low-temperature disruptor. Centrifuge at 8000 rpm for 30 minutes, and collect the supernatant as the crude enzyme solution.

[0102] (6) Protein concentration measurement of crude enzyme solution: The total protein content in the crude enzyme solution was measured using a Pierce BCA protein quantification kit in a multifunctional microplate reader.

[0103] Example 4: Univariate Factor Exploration of Glycosyltransferases

[0104] A single variable factor exploration experiment of the enzyme catalytic reaction conditions was conducted on the glycosyltransferase (shown as SEQ ID NO.1) to identify its enzyme properties.

[0105] 1) To study the effect of reaction system pH on the catalytic efficiency of glycosyltransferases, three buffer systems, acetate buffer (50 mM, pH buffer range 4.0-5.5), phosphate buffer (50 mM, pH buffer range 6.0-8.0), and Tris hydrochloride buffer (50 mM, pH buffer value 7.5-9.0), were used at 32.5°C. Three corresponding reaction systems were then prepared (see Table 1), and changes in enzyme activity within the pH range of 4.0-9.0 were detected.

[0106] As shown in Figure 1, the optimum pH of the enzyme is 8.0, and when the pH is equal to 6.0, the catalytic efficiency can be maintained at 65%, indicating that the enzyme has certain potential for large-scale biocatalytic production of rebaudioside M. The optimum pH and catalytic efficiency of the remaining amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 are similar to those of the amino acid sequence shown in SEQ ID NO.1, as shown in Figure 1.

[0107] 2) To investigate the effect of temperature on the catalytic efficiency of glycosyltransferase, the catalytic efficiency of the enzyme was tested at 16°C, 22°C, 30°C, 32.5°C, 37°C, 42°C, and 65°C at pH 8.0. The catalytic efficiency of the glycosyltransferase decreased significantly at temperatures above 40°C, but it exhibited higher catalytic activity between 30°C and 37°C (as shown in Figure 2).

[0108] As shown in Figure 2 , the yield of rebaudioside M was highest at 37° C., thus, this enzyme (the trend was consistent for all glycosyltransferases) had the highest catalytic efficiency at 37° C. The optimal temperatures and catalytic activities of the remaining amino acid sequences shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 were similar to those of the amino acid sequence shown in SEQ ID NO. 1, as shown in Figure 2 .

[0109] 3) This experiment investigated the effects of various metal ions and ammonium ions on the catalytic efficiency of glycosyltransferases. As shown in Figure 3, Mg 2+ , Ca 2+ 、Mn 2+ and Fe 3+ The catalytic efficiency of the enzyme is significantly promoted, among which Mg 2+ The promoting effect of Ca was the strongest. Under the same conditions, the yield of rebaudioside M could reach 61%, which was about 6 times higher than that of the blank control group. 2+ and Mn 2+The yield of rebaudioside M under the condition of presence of NH 4+ It also has a strong promoting effect on the enzyme, and the yield of rebaudioside M is 55%. 2+ 、Zn 2+ and Ni 2+ It has a significant inhibitory effect on the catalytic efficiency of the enzyme, among which Cu 2+ The inhibitory effect of Zn on the enzyme was the strongest. In the presence of copper ions, the enzyme lost its activity and the yield of rebaudioside M was 0. 2+ In the presence of β-catenin, the catalytic efficiency of the enzyme was only half of that of the blank control group.

[0110] The above results indicate that the enzyme is a metal-dependent enzyme, in which Mg 2+ The most effective way to promote the enzyme is Cu 2+ Ions have the strongest inhibitory effect on the enzyme and can directly destroy the enzyme protein structure and thus make it lose its activity.

[0111] The effects of different metal ions and ammonium ions on the catalytic efficiency of the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 are similar to those of the amino acid sequence shown in SEQ ID NO.1, as shown in Figure 3.

[0112] Example 5: Synthesis of Rebaudioside M from Rebaudioside A by Dual Enzyme Method Using Sucrose as Glycosyl Donor

[0113] 1) Catalytic reaction system

[0114] Add 1 mM UDPG (uridine diphosphate glucose), crude glycosyltransferase enzyme (~3 mg / mL total protein), 1 g / L rebaudioside A, and 5 mM magnesium chloride to the reaction system. Supplement with 50 mM PBS (pH 8.0) to the final reaction volume. Incubate at 37°C, 200 rpm for 2 hours. Samples are then heated in a 100°C water bath for 10 minutes. Centrifuge at 12,000 rpm for 1 minute at room temperature. Filter the supernatant through a 0.45 μm sterile filter membrane into a liquid phase vial for analysis by HPLC.

[0115] 2) HPLC detection method

[0116] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the enzymatic reaction system. The chromatographic column model was an Inertsil™ ODS-3 reverse-phase C18 column; the column temperature was 40°C; the detector was a PDA (ultraviolet) detector; the detection wavelength was 218 nm; the mobile phase A was acetonitrile containing 0.1% formic acid; the mobile phase C was ultrapure water containing 0.1% formic acid, where % is the volume percentage; the liquid phase gradient elution program was: 0 min (25% A, 75% C), 4 min (40% A, 60% C), 15 min (42% A, 58% C), 16 min (100% A, 0% C), 21 min (5% A, 95% C), 30 min (25% A, 75% C); the flow rate was 0.6 mL / min; and the injection volume was 10 μL.

[0117] 3) Results

[0118] As shown in FIG4 , according to the analysis and calculation based on the liquid phase detection results, after 2 h of reaction, rebaudioside A was almost completely converted into rebaudioside M, with a conversion rate of up to 98%, and no by-products were generated.

[0119] Example 6: Enzymatic synthesis of rebaudioside M from rebaudioside A using UDPG (uridine diphosphate glucose) as a glycosyl donor

[0120] 1) Catalytic reaction system

[0121] Add 34 g / L sucrose, 10 g / L rebaudioside A, crude glycosyltransferase enzyme solution (~5 mg / mL total protein), crude sucrose synthase enzyme solution (~2 mg / mL total protein), 2 mM uridine diphosphate disodium salt, and 5 mM magnesium chloride to the reaction system. Supplement with 50 mM PBS (pH 8.0) buffer to the final reaction volume. Incubate at 37°C, 200 rpm for 3 h. Samples were heated in a 100°C water bath for 10 min. Centrifuge at 12,000 rpm for 1 min at room temperature. The supernatant was filtered through a 0.45 μm sterile filter membrane into a liquid phase vial and analyzed by HPLC.

[0122] 2) HPLC detection method

[0123] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the enzymatic reaction system. The chromatographic column model was an Inertsil™ ODS-3 reverse-phase C18 column; the column temperature was 40°C; the detector was a PDA (ultraviolet) detector; the detection wavelength was 218 nm; the mobile phase A was acetonitrile containing 0.1% formic acid; the mobile phase C was ultrapure water containing 0.1% formic acid, where % is the volume percentage; the liquid phase gradient elution program was: 0 min (25% A, 75% C), 4 min (40% A, 60% C), 15 min (42% A, 58% C), 16 min (100% A, 0% C), 21 min (5% A, 95% C), 30 min (25% A, 75% C); the flow rate was 0.6 mL / min; and the injection volume was 10 μL.

[0124] 3) Results

[0125] As shown in FIG5 , based on the analysis and calculation of the liquid phase detection results, after 3 h of the dual-enzyme catalysis of rebaudioside A using sucrose as the glycosyl donor, rebaudioside A was almost completely converted into rebaudioside M, with a conversion rate of up to 95%, and no by-products were generated.

[0126] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for efficiently preparing rebaudioside M using a bioenzymatic method, characterized in that: The steps include: Rebaudioside A, uridine diphosphate glucose, glycosyltransferase and salt are added into a buffer solution, mixed evenly to obtain a mixed solution, and an enzyme-catalyzed reaction is carried out under stirring to obtain the rebaudioside M.

2. The method for efficiently preparing rebaudioside M using a biological enzyme method according to claim 1, characterized in that: In the mixed solution, the concentration of rebaudioside A is 1g / L-100g / L; in the mixed solution, the concentration of uridine diphosphate glucose is 0.5mM-20mM; the salt is one or more of calcium salt, cobalt salt, iron salt, magnesium salt, manganese salt, ammonium salt, nickel salt, and zinc salt; in the mixed solution, the concentration of the salt is 1-10mM.

3. The method for efficiently preparing rebaudioside M using a biological enzyme method according to claim 2, characterized in that: The salt is one or more of calcium salt, magnesium salt, manganese salt and iron salt; in the mixed solution, the concentration of the salt is 0.1-5mM.

4. The method for efficiently preparing rebaudioside M by using a bioenzymatic method according to claim 1, characterized in that: In the mixed solution, the pH of the buffer solution is 5.0-9.0; the stirring rate under stirring is 50-220 rpm; the temperature of the enzyme catalytic reaction is 20-45° C., and the time of the enzyme catalytic reaction is 1-5 h.

5. The method for efficiently preparing rebaudioside M using a biological enzyme method according to claim 1, characterized in that: In the mixed solution, the concentration of the glycosyltransferase is 0.1-20 g / L; the sequence of the glycosyltransferase is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.

12.

6. A method for efficiently preparing rebaudioside M using a bioenzymatic method, characterized in that: The steps include: Rebaudioside A, sucrose, 5-uridine diphosphate disodium salt, glycosyltransferase, sucrose synthase and salt are added to a buffer solution, mixed evenly to obtain a mixed solution, and an enzyme-catalyzed reaction is carried out under stirring to obtain the rebaudioside M.

7. The method for efficiently preparing rebaudioside M by using a biological enzyme method according to claim 6, characterized in that: In the mixed solution, the concentration of rebaudioside A is 1-100 g / L; in the mixed solution, the concentration of sucrose is 1-500 mM; in the mixed solution, the concentration of 5-uridine diphosphate disodium salt is 0.1-20 mM; the salt is one or more of calcium salt, cobalt salt, iron salt, magnesium salt, manganese salt, ammonium salt, nickel salt and zinc salt; in the mixed solution, the concentration of salt is 0.1-10 mM; the pH of the buffer solution is 5.0-9.0; the stirring rate in the stirring state is 50-220 rpm; the temperature of the enzyme catalyzed reaction is 20-45° C., and the time of the enzyme catalyzed reaction is 1-10 h.

8. The method for efficiently preparing rebaudioside M by using a biological enzyme method according to claim 7, characterized in that: The salt is one or more of calcium salt, magnesium salt, manganese salt and iron salt; in the mixed solution, the concentration of the salt is 0.1-5mM; the pH of the buffer solution is 6.0-8.0; and the temperature of the enzyme catalytic reaction is 30-37°C.

9. The method for efficiently preparing rebaudioside M by using a biological enzyme method according to claim 6, characterized in that: In the mixed solution, the concentration of the glycosyltransferase is 0.1-20 g / L; the sequence of the glycosyltransferase is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.

12.

10. The method for efficiently preparing rebaudioside M by using a biological enzyme method according to claim 6, characterized in that: In the mixed solution, the concentration of the sucrose synthase is 0.1-10 g / L; the sequence of the sucrose synthase is one or more of the amino acid sequences shown in SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20.

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