A glycosyltransferase and its use in producing glycosides

Abstract

The present invention discloses a glycosyltransferase and its application in the production of glycosides. The present invention clones a glycosyltransferase from a low-temperature resistant Bacillus subtilis using molecular cloning technology, constructs an expression vector with the cloned glycosyltransferase molecule and successfully expresses it in vitro. The enzyme activity test shows that the glycosyltransferase expressed in vitro can catalyze rebaudioside A to rebaudioside D, and the glycosyltransferase has the characteristic of low-temperature resistance, can still catalyze the reaction under the condition of 25°C, has a wide pH value reaction range, and can achieve the catalytic reaction within the range of pH6-8. The new glycosyltransferase disclosed in the present invention has the advantages of low-temperature resistance and wide pH value reaction, and is suitable for the production of various glycoside glycosylation products.

Description

Technical Field

[0001] The invention relates to the fields of gene cloning and molecular biology, and in particular to a glycosyltransferase and an application thereof in producing glycosides. Background Art

[0002] The enzymes involved in catalyzing the glycosylation of compounds in plants are called glycosyltransferases (GTs), which transfer active sugar donors to sugar acceptors to form acceptor glycosylation products. UDPG-glycosyltransferases (UGTs) are a type of glycosyltransferase that depends on the UDPG (uridine diphosphate) sugar donor.

[0003] Glycosyltransferases (EC2.4.XY) are the most diverse class of enzymes in nature. Glycosyltransferases are responsible for transferring the monosaccharide part of the active donor to sugars, proteins, lipids, nucleic acids and other small molecules, that is, completing the glycosylation reaction to form glycosylated products with many biological functions. There are a large number of glycosyltransferases in prokaryotes and eukaryotes, which are used to catalyze glycosylation reactions to generate oligosaccharides, polysaccharides and sugar complexes. These structurally diverse sugar molecules participate in and mediate life activities from structure, storage to signal transmission. Glycosyltransferases can transfer the sugar group on the activated glycosyl donor to a specific receptor molecule to form a glycosidic bond, and during the transfer of the sugar group, the configuration of the anomeric carbon atom is retained or flipped to form glycosylated products of different configurations.

[0004] UGTs belong to the GT1 multigene family, which accounts for about half of the entire GTs family. The proteins encoded by this type of gene usually have a conserved sequence of 44 amino acids at the C-terminus, which may be involved in binding UDPG in the glycosylation reaction and is also called the PSPG box. In the plant multigene GTs family, they are generally involved in various plant growth regulation and adverse stress.

[0005] Glycosyltransferases play an important biological role in all living organisms and are increasingly attracting people's attention. Protein glycosylation modification and deglycosylation of natural glycoproteins are one of the important means to study the structure and function of glycoprotein sugar chains. Glycosyltransferases are useful tools in this reaction.

[0006] Glycosyltransferase (GT) is a widely existing transferase in organisms that can transfer the sugar on the activated glycosyl donor to the substrate acceptor. Most glycosyl donors are nucleotide sugars, but sugar phosphates, glycolipids and other sugars can also be donors. The types of glycosyl acceptors include proteins, sugars, lipids, nucleic acids and various secondary metabolites such as antibiotics, alkaloids, plant hormones, plant polysaccharides, etc.

[0007] In the past few decades, a lot of research has focused on exploring the importance of glycosyltransferases in organisms. Limited biochemical data have restricted further understanding of the functions of specific single enzymes. In recent years, the identification of GTs genes, biochemical characterization, and their analysis at the transcriptional and metabolic levels in organisms have facilitated the study of glycosylation mechanisms. Large-scale plant genome sequencing has helped to decipher the functions of GTs in the biosynthesis of glycoconjugates during plant secondary metabolism.

[0008] Plant GTs participate in the biosynthesis of sugar complexes with different characteristics, transferring sugar moieties to aglycones or branched sugar moieties, thereby forming a broad spectrum of secondary specific metabolites. Plants produce a large number of natural products with great application value in the pharmaceutical and food industries through secondary metabolic processes.

[0009] As a key modification step in biological processes, glycosylation mechanisms contribute to the formation of complex and diverse plant secondary metabolites. Since most high-value secondary metabolites are not naturally present in high levels, direct extraction from natural plants usually results in low yields and high energy consumption. In addition, the benefits of natural extraction may be highly dependent on geographical, seasonal, and even political factors. In vitro plant cell culture or metabolic engineering of natural plants can solve the problems of seasonal factors and low yields to a certain extent, but at a high economic cost. There are many types of natural glycosides, and most of them have stereoselectivity for specific glycosidic bonds, which makes them affected by many reactive groups. Therefore, it is difficult to obtain large quantities of products through chemical synthesis.

[0010] Therefore, driven by the high demand for high-value natural products and the need for sustainable development, biosynthetic methods have emerged. Among them, GT catalysis has high selectivity and high catalytic efficiency, which can achieve stereoselectivity and regioselectivity in branch-extended glycosylation of macromolecular glycans and sugar conjugates, and has been successfully applied in the synthesis of glycoside molecules in various metabolites.

[0011] The synthesis of glycosides is of great significance in the pharmaceutical, cosmetic, food and health care industries. In particular, the selective glycosylation of complex substrates is a major challenge in chemical synthesis, requiring a multi-step and complex synthesis process. Glycosyltransferases, due to their special regioselectivity and stereoselectivity, can perform a one-step glycosylation reaction on specific positions of complex compounds without the need for group protection in chemical synthesis. In addition, glycosyltransferases also have a wide spectrum of substrates, which makes them have higher application value. However, the high cost and low availability of nucleoside diphosphate sugars limit the application of glycosyltransferases in industry.

[0012] Stevia rebaudiana Bertoni is a perennial rhizome shrub in the Asteraceae family, native to Paraguay and Brazil, and has been used to optimize the flavor of tea for hundreds of years. There are 220-230 related stevia species in this genus. Steviosides (SGs) are a general term for diterpene glycoside components extracted from stevia leaves and are the source of sweetness in stevia. Based on a large amount of beneficial evidence in recent years, stevioside has now been approved by the US Food and Drug Administration (FDA), the Joint FAO / WHO Expert Committee on Food Additives (JECFA) and the European Food Safety Authority as a food that can be consumed by humans. With more and more stevioside products on the market, the European Food Safety Authority has analyzed the intake of steviosides by people of different ages in different European countries in the past few years. According to the analysis reports in 2015 and 2018, the trend of using stevia ingredients in daily diet is increasing. In the next few years, the global market for stevia sweeteners is expected to grow to millions of tons.

[0013] In addition to its sweet taste, stevioside has also received attention in clinical research. Current studies have found that stevioside has important pharmacological and therapeutic activities, such as antioxidant, antibacterial, antifungal, antiviral, antitumor, diuretic, stomach protection and antidiarrheal functions, and has a positive effect on immune regulation and kidney function, blood pressure and blood sugar. Stevioside is added to the daily diet as a sweetener, which is very beneficial for patients with metabolic syndrome, type II diabetes, hypertension, cardiovascular disease, hypoglycemia, dental caries and candidiasis. And stevia itself can be used as a dressing for wound healing and skin abrasions. Current experimental results show that stevioside is non-carcinogenic, non-genotoxic and has nothing to do with human reproductive and developmental toxicity.

[0014] Currently, all stevioside products on the market have a slight bitter and astringent taste, and a metallic aftertaste that cannot be concealed by human taste buds, which limits the application of stevioside in food and pharmaceutical products. In order to minimize the bitter aftertaste, a variety of industrial methods have been adopted, such as using maltodextrin and inulin as capsules and spray drying technology to improve their flavor. At the same time, the aftertaste can also be effectively concealed by compounding other sweeteners, such as erythritol, maltitol, inulin, oligofructose, etc.

[0015] The structure of different types of steviol glycosides also determines their sweetness and aftertaste. The glycosyl residues at C-19 (R 1) and C-13 (R 2) of steviosides affect the sweetness. Stevioside (ST) and rebaudioside A (Reb A) are the two most common steviol glycosides on the market, and Reb A has a higher sweetness than ST. Compared with ST, steviolbioside has only one less glucosyl group at R1, but its sweetness is lower than ST. By analyzing the R1 group of C13 and comparing RebA and rubusoside, it can be found that the change of glycosyl on the R1 group may be related to the aftertaste of stevioside. Extending the glycosyl aglycone on the C19 side chain can increase the sweetness of stevioside. Among them, rebaudioside D (Reb D) has a relatively low aftertaste compared with other steviol glycosides.

[0016] Among the glycosides extracted from stevia, stevioside (ST) and rebaudioside A

[0017] (Rebaudioside A, Reb A) are the two ingredients with the highest content. Among them, the content of Reb A is lower than that of ST, but its bitterness is less than that of ST, and its stability is also higher, which is more advantageous in practical applications. Since 2008, RebA has passed the safety certification of the US Food and Drug Administration and is allowed to be used as a food additive. After December 2011, ST was also allowed to be sold and used as a food additive in the European Union. At present, in my country, many types of food and beverages have used steviol glycoside products to replace sucrose. Increasing the content of Reb A in steviol glycoside products is conducive to expanding the scope of application of stevia products and meeting market demand.

[0018] Although in previous studies, it was predicted that Reb A would be the sweetest stevioside. The flavors of Reb A, Reb D and Reb M were subjectively evaluated by volunteers. The experiment showed that the sweetness of Reb A was significantly lower than that of Reb M, and when actually tasted, the sweetness was slightly lower than that of Reb D but there was no significant difference. However, in comparison, the bitterness of Reb A was very prominent in the sample, while Reb D and Reb M did not show a strong aftertaste compared with sucrose. Therefore, Reb D and Reb M, as a new generation of sweeteners, are more popular with consumers and have broader application prospects.

[0019] Purified glycosyltransferases are important enzymes in glycosylation engineering, providing an excellent method for studying the relationship between the structure and function of glycoprotein sugar chains and cell surface glycosylation modification. Currently, research on these enzymes is still in its infancy, and there are still very few purified enzymes. Summary of the invention

[0020] The purpose of the present invention is to provide a new glycosyltransferase to solve the problem of insufficient glycosyltransferase in the prior art, especially the lack of characteristic glycosyltransferase, which restricts the industrial production of various types of stevioside products.

[0021] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0022] The invention discloses a glycosyltransferase, and the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO.4.

[0023] The invention discloses a nucleotide molecule encoding the glycosyltransferase.

[0024] Preferably, the sequence of the nucleotide molecule is as shown in SEQ ID NO.3.

[0025] Preferably, the glycosyltransferase is from Bacillus subtilis.

[0026] The invention discloses an expression vector, which contains the nucleotide molecule.

[0027] The invention discloses a cell, which comprises the expression vector.

[0028] Preferably, the cell is a eukaryotic cell or a prokaryotic cell.

[0029] Preferably, the cell is yeast or Escherichia coli.

[0030] The invention discloses glycosyltransferase, which is obtained by expressing the cell in vitro.

[0031] The present invention discloses the use of the glycosyltransferase and / or the nucleotide molecule and / or the expression vector and / or the cell in producing glycoside.

[0032] The invention discloses a method for producing glycosides, which is characterized in that the glycosyltransferase is used for production.

[0033] The invention discloses a method for producing glycosides, comprising an operation of catalyzing rebaudioside A to rebaudioside D, wherein the amino acid sequence of the glycosyltransferase used in the operation is shown in SEQ ID NO.4.

[0034] Preferably, the temperature of the catalytic reaction is 25°C.

[0035] The present invention clones a glycosyltransferase from a low-temperature resistant Bacillus subtilis by using molecular cloning technology, constructs an expression vector with the cloned glycosyltransferase molecule and successfully expresses it in vitro. The enzyme activity test shows that the glycosyltransferase expressed in vitro can catalyze rebaudioside A to rebaudioside D, and the glycosyltransferase has the characteristics of low-temperature resistance, can still catalyze the reaction at 25°C, has a wide pH value reaction range, and can achieve the catalytic reaction in the pH range of 6-8. The new glycosyltransferase disclosed in the present invention has the advantages of low-temperature resistance and wide pH value reaction, and is suitable for the production of various glycoside glycosylation products. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 .Gel electrophoresis results of PCR-amplified glycosyltransferase of Bacillus subtilis, where lanes 1 and 2 are PCR-amplified products and lane 3 is a negative control.

[0037] Figure 2 .PCR amplification and identification results of the constructed expression vector GT-PET-28A.

[0038] Figure 3 .Double enzyme digestion identification results of the constructed expression vector GT-PET-28A.

[0039] Figure 4 .SDS-PAGE electrophoresis of expressed target protein after bacterial cell disruption;

[0040] Figure 5 .SDS-PAGE electrophoresis of the expressed target protein after purification by nickel column;

[0041] Figure 6 .The results of HPLC detection of the enzymatic reaction products, among which, Figure 6 a is the detection result of the reaction product, Figure 6 b is the detection result of the reaction solution containing only rebaudioside A (Reb A), Figure 6 c is the detection result of the prepared reaction solution containing only rebaudioside D (Reb D).

[0042] Figure 7 . The low temperature enzymatic reaction product was tested by HPLC, among which, Figure 7 a is the detection result of the reaction product, Figure 7 b is the detection result of the reaction solution containing only rebaudioside A (Reb A), Figure 7 c is the detection result of the prepared reaction solution containing only rebaudioside D (Reb D).

[0043] Figure 8 .Detection results of enzymatic reaction products under different pH reaction conditions. DETAILED DESCRIPTION

[0044] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0045] Example 1 Cloning and sequencing of glycosyltransferase genes

[0046] The genomic DNA of the low-temperature resistant Bacillus subtilis was extracted as an amplification template. The low-temperature resistant Bacillus subtilis strain was screened and preserved for this experiment. The strain can still grow and reproduce at a temperature as low as 10°C, which is smaller than that of the common Bacillus subtilis at normal temperature.

[0047] According to the sequence information of the glycosyltransferase gene of Bacillus subtilis published in NCBI, the amplification primer sequences were designed according to its conserved segments as follows:

[0048] Upstream primer: 5'-atgaaaaagcaccatattt-3' (SEQ ID NO. 1);

[0049] Downstream primer: 5'-ttattgcggtacagcgga-3' (SEQ ID NO. 2).

[0050] Using the extracted low-temperature-resistant Bacillus subtilis genome as a template, a PCR amplification system was established as follows:

[0051] 10XPCR reaction buffer 2μL,

[0052] 1μmol / L positive upstream and downstream primers,

[0053] 1U Taq DNA enzyme,

[0054] Template DNA 1 μL,

[0055] The reaction volume was made up to 20 μL.

[0056] The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 5 min, cyclic amplification at 94°C for 1 min, 58°C for 1.5 min, and 72°C for 3 min (30 cycles), and extension at 72°C for 5 min. Figure 1 ), the size of the detected amplified fragment was similar to the size of the glycosyltransferase of Bacillus subtilis that has been published. The PMD-T vector was recovered and sequenced, and the sequencing results are shown in the following sequence 3.

[0057] Sequenced sequence of the cloned glycosyltransferase (SEQ ID NO.3): atgaaaaagc accatatttcgatgatcaat atccctgcgt acgggcatgt caatcctacgctagcattag tggagaagct ttgtgagaaagggcatcgtg tcacgtatgc gacgactgaggaatttgcgc ccgctgttca gcaagccggt ggagaagcattgatttatca tacatccttgaatattgatc ctaagcaaat cagggagatg atggaaaaga atgacgcgacgctcagtctattgaaagaat cactcagcat tctgccgcag cttgaggagt tatataaagatgatcagcctgatctgatca tctatgactt tgtcgcactt gcgggaaaat tgtttgctgataaacttaatgtgccggtca tcaagctctg ttcatcatat gcccaaaatg aatcctttcagcttggaaatgaagacatgc tgaaaaagat aaaagaagcc gaggctgaat ttaaagcctacttggagcaagagcaattgc cggctgtttc atttgaacaa ttagctgtgc cggaagcattaaatattgtctttatgccga aatcctttca gattcagcat gagacgttcg atgaccgtttctgttttgtcggcccttccc ttggaaaacg gacggaacaa gaaagcctgt tgattgacaagggtgatcgtgcgcttatgc tgatttcttt gggaacggca tttaacgcat ggccggaattttacaagatgtgcatcgatg catttcggga ttcttcatgg caagtgatca tgtcggtcgggaaatcgattcatcctgaaa gcttggatga tacccctgct aactttacca ttcgccaaagcgtgccgcatcttgaggtgt tagcgaaagc cgatttgttt atttctcatggcgggatgaacagtacgatggaagcgatga atgccggtgt gccgctcgtc gtcattccgc aaatgtatgagcaggagctcaccgcaaagc gtgtcgatga gttaggtctt ggcgtttatttgcaaagagaagaagttcctgtttccaagc tgcaggaagc ggttcaggcc gtatccggtg atcaagagctgctcagccgcgtcaagagta tgcaaaagga tgtaaaagaa gcaggcggag cggagcgtgcggcagctgagattgaagcgt ttatgaaaaa atccgctgta ccgcaataa.

[0058] The corresponding encoded amino acid sequence is as follows (SEQ ID NO.4):

[0059] MKKHHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGGEALIYHTSLNIDPKQ

[0060] IREMMEKNDATLSLLKESLSILPQLEELYKDDQPDLIIYDFVALAGKLFADKLNVPVIKLCSSYAQ

[0061] NESFQLGNEDMLKKIKEAEAEFKAYLEQEQLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDRFC

[0062] FVGPSLGKRTEQESLLIDKGDRALMLISLGTAFNAWPEFYKMCIDAFRDSSWQVIMSVGKSIHPES

[0063] LDDTPANFTIRQSVPHLEVLAKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTAKRVDELGLGVYLQREEVPVSKLQEAVQAVSGDQELLSRVKSMQKDVKEAGGAERAAAEIEAFMKKSAVPQ.

[0064] Example 2 Prokaryotic expression and purification of glycosyltransferase

[0065] 2.1 Cloning of glycosyltransferase genes and construction of expression vectors

[0066] According to the detected glycosyltransferase sequence, expression primers were designed, and BamH I and Xho I restriction sites were introduced at both ends of the designed primers respectively:

[0067] Upstream primer: 5'-ggatccatgaaaaagcaccatat-3' (SEQ ID NO. 5);

[0068] Downstream primer: 5'-ctcgagttattgcggtacagcg-3' (SEQ ID NO. 6).

[0069] The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 5 min, cyclic amplification at 94°C for 1 min, 58°C for 1.5 min, and 72°C for 3 min (30 cycles), and extension at 72°C for 5 min.

[0070] After the amplified product was connected to the PMD-18T vector, it was transformed into DH5α competent cells, and the positive clones were selected for amplification and culture. The positive recombinant plasmid was extracted and double-digested with BamHI and Xho I. The double-digestion product was recovered and connected with PET-28A that was also digested with the same enzymes to transform DH5α. The positive recombinant plasmid GT-PET-28A was identified by PCR and double-digestion, and then transformed into BL21 (DE3) competent cells. The positive clones were selected for culture. The results of PCR and double-digestion identification are shown as follows: Figure 2-Figure 3 .

[0071] 2.2 Inducible expression and purification of glycosyltransferases

[0072] The BL21 (DE3) engineering bacteria containing GT-PET-28A were inoculated and cultured overnight. The cells were transferred to a new ampicillin LB medium at a ratio of 1 / 1000. When the OD value reached between 0.6 and 0.8, IPTG was added for induction. After IPTG induction, SDS-PAGE was performed for identification. After Coomassie Brilliant Blue staining, an obvious specific protein band was found at about 47KD. The results of SDS-PAGE electrophoresis can be found in Figure 4 .

[0073] According to the HIS tag characteristics of the PET-28A vector, the crude enzyme solution was purified using a nickel column. After SDS-PAGE gel identification, an obvious specific band was found at 47KD. The electrophoresis image of the purified glycosyltransferase protein can be found in Figure 5 .

[0074] Example 3 Identification of glycosyltransferase-catalyzed substrates

[0075] Establish an enzyme catalytic reaction system, including:

[0076] UDP-glucose (UDPG) 2.5 mM / L;

[0077] Purified glycosyltransferase protein 25 μg / μL;

[0078] Candidate catalytic substrates, 0.2 mM: rebaudioside A (Reb A);

[0079] The buffer system of the reaction was: 100 mM / L pH 7.5 Tris-HCl;

[0080] β-Mercaptoethanol (volume ratio): 0.1%;

[0081] The reaction system was a 50 μL enzyme reaction system, and the reaction conditions were: the reaction was carried out in a 37°C water bath for 30 minutes, and the reaction was terminated by adding 50 μL of chromatographically pure methanol, and then stored at -20°C for HPLC detection.

[0082] HPLC detection conditions for stevioside:

[0083] Chromatographic column: Phenomenex Gemini C18 reverse phase column (250 mm × 4.6 mm);

[0084] The column temperature was 42°C;

[0085] Mobile phase A was 50 mM Tris-HCl buffer.

[0086] Mobile phase B was acetonitrile;

[0087] The flow rate was 1 mL / min; the injection volume was 10 μL; and the detection wavelength was 262 nm.

[0088] Turn on the liquid chromatograph and workstation. Flush the system with acetonitrile until the pressure is stable, turn on the liquid chromatograph and workstation. First flush the system with 1mL / min acetonitrile until the pressure is stable, reduce the flow rate to 0.5mL / min, connect the chromatographic column and flush until the pressure is stable. Adjust the mobile phase to 80% ultrapure water and 20% acetonitrile, flush until the system pressure is stable. Adjust the mobile phase to 65% mobile phase A and 35% mobile phase B, flush until the system pressure is stable, and then perform sample testing. For test results, see Figure 6 .

[0089] After the sample test is completed, the mobile phase is adjusted to 80% ultrapure water and 20% acetonitrile, and the system pressure is flushed until it stabilizes. Flush the system with 1mL / min acetonitrile until the pressure stabilizes, reduce the flow rate to 0.5mL / min, remove the chromatographic column and flush until the pressure stabilizes.

[0090] Figure 6 The test results showed that the glycosyltransferase gene cloned from Bacillus subtilis, after in vitro expression and purification, could catalyze the glycosylation of rebaudioside A (Reb A) to produce rebaudioside D (Reb D).

[0091] Example 4 Identification of the reaction temperature of glycosyltransferase-catalyzed substrate

[0092] Considering that the prepared glycosyltransferase is derived from the low-temperature-resistant Bacillus subtilis, which can carry out normal physiological metabolism at low temperatures, we speculate that the prepared glycosyltransferase may also have the property of low-temperature resistance.

[0093] Based on the reaction system of Example 3, the reaction temperature was adjusted to 25° C. for enzymatic reaction.

[0094] The specific operations are as follows:

[0095] Establish an enzyme catalytic reaction system, including:

[0096] UDP-glucose (UDPG) 2.5 mM / L;

[0097] Purified glycosyltransferase protein 25 μg / μL;

[0098] Candidate catalytic substrates, 0.2 mM: rebaudioside A (Reb A);

[0099] The buffer system of the reaction was: 100 mM / L pH 7.5 Tris-HCl;

[0100] β-Mercaptoethanol (volume ratio): 0.1%;

[0101] The reaction system was a 50 μL enzyme reaction system, and the reaction conditions were: the reaction was carried out in a 25°C water bath for 30 minutes, and the reaction was terminated by adding 50 μL of chromatographically pure methanol, and then stored at -20°C for HPLC detection.

[0102] Test results see Figure 7 From the test results, adjusting the enzyme reaction system to 25°C can achieve the same reaction as 37°C, indicating that the glycosyltransferase obtained in the present invention is a low-temperature resistant type and can still maintain enzyme activity at a lower temperature.

[0103] Example 5 Identification of pH of glycosyltransferase-catalyzed substrate reaction

[0104] The appropriate reaction pH value of the purified glycosyltransferase was tested as follows:

[0105] Establish an enzyme catalytic reaction system, including:

[0106] UDP-glucose (UDPG) 2.5 mM / L;

[0107] Purified glycosyltransferase protein 25 μg / μL;

[0108] Candidate catalytic substrates, 0.2 mM: rebaudioside A (Reb A);

[0109] The buffer system for the reaction was: 100 mM / L Tris-HCl with pH values ​​of 6.0, 7.0, 8.0, and 9.0;

[0110] β-Mercaptoethanol (volume ratio): 0.1%;

[0111] The reaction system is a 50 μL enzyme reaction system, and the reaction conditions are: the reaction is carried out in a 37°C water bath for 60 minutes, and the reaction is terminated by adding 50 μL of chromatographic pure methanol, and then stored at -20°C for HPLC detection. Three parallel reactions are carried out for each pH value. The peak area detected is used to reflect the product of the enzymatic reaction. The test results are shown in Figure 8 , indicating that the optimum pH value of the reaction of the purified glycosyltransferase of the present invention is about 8.0, and the catalytic reaction can be achieved at pH 6-8. After the pH value reaches 9, the enzyme reaction activity decreases rapidly.

[0112] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A glycosyltransferase, characterized in that The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO.

4.

2. A nucleotide molecule encoding the glycosyltransferase according to claim 1.

3. The nucleotide molecule according to claim 2, characterized in that The sequence of the nucleotide molecule is shown in SEQ ID NO.

3.

4. The glycosyltransferase according to claim 1, characterized in that The glycosyltransferase is from Bacillus subtilis.

5. An expression vector, characterized in that The expression vector comprises the nucleotide molecule according to claim 2 or 3.

6. A cell, characterized in that The cell comprises the expression vector of claim 5.

7. The cell according to claim 6, characterized in that The cell is a eukaryotic cell or a prokaryotic cell.

8. The cell according to claim 7, characterized in that The cell is yeast or Escherichia coli.

9. A glycosyltransferase, characterized in that The glycosyltransferase is obtained by expressing the cell according to any one of claims 6 to 8 in vitro.

10. Use of the glycosyltransferase according to any one of claims 1, 4, and 9 and / or the nucleotide molecule according to any one of claims 2-3 and / or the expression vector according to claim 5 and / or the cell according to any one of claims 6-8 in catalyzing the production of rebaudioside D from rebaudioside A.

11. A method for catalyzing rebaudioside A to produce rebaudioside D, characterized in that: The method is carried out using the glycosyltransferase described in any one of claims 1, 4 and 9.

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