Application of TB culture medium in soluble expression level of glycosyl transferase UGT91D2

Abstract

The invention discloses an application of a TB culture medium in the soluble expression level of glycosyl transferase UGT91D2, and the TB culture medium realizes the improvement of the soluble expression quantity of recombinant UGT91D2 protein by maintaining the pH stability of a cell growth process in an escherichia coli E.coli BL21 expression system and improving the thallus density.

Description

Technical Field

[0001] This invention belongs to the fields of protein engineering and fermentation engineering technology, specifically relating to the application of TB culture medium in the soluble expression level of glycosyltransferase UGT91D2. Background Technology

[0002] Steviol glycosides (SGs) extracted from stevia leaves are low in calories and high in sweetness, and have been widely used in the food industry as natural sweeteners. With the development of genome sequencing technology, researchers have revealed the biosynthetic mechanism of steviol glycosides, a process involving multiple glycosyltransferases.

[0003] Among them, the glycosyltransferase UGT91D2 catalyzes the formation of a 1,2-β-D-glycosidic bond on the C13-glycosyl group of rub to generate steviol glycoside (St). Simultaneously, this enzyme can also catalyze the glycosylation reaction of the C19 glucose group of St and Reb A to generate Reb E and Reb D, respectively. From a comprehensive evaluation of sweetness and aftertaste, Reb D showed the best performance. However, unfortunately, compared with other glycosyltransferases, research on the catalytic characteristics, enzymatic mechanism, and application potential of UGT91D2 in the synthesis of steviol glycosides is still very limited and has not been fully explored.

[0004] To quantitatively analyze the enzymatic properties of UGT91D2 and assess its application potential in the glycosylation pathway, obtaining a soluble and active target protein is essential. However, the recombinant expression and preparation of this enzyme face significant challenges. Previous studies have reported that attempts to express UGT91D2 glycosyltransferase in eukaryotic expression systems (such as yeast) have encountered severe degradation by endogenous proteases, resulting in extremely low or even no yield of the target protein. While cell-free translation systems using wheat germ can synthesize the protein, it lacks activity. These difficulties severely hinder research on the enzymatic properties, substrate specificity, and catalytic efficiency of the glycosyltransferase UGT91D2. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an application of TB culture medium in the soluble expression level of glycosyltransferase UGT91D2.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the application of TB culture medium in the soluble expression level of glycosyltransferase UGT91D2, characterized in that: the TB culture medium maintains pH stability and increases cell density during cell growth in the E. coli BL21 expression system, thereby increasing the soluble expression level of recombinant UGT91D2 protein.

[0009] In a preferred embodiment of the application described in this invention, the culture temperature of the TB culture medium is 16-25℃.

[0010] In a preferred embodiment of the application described in this invention, the culture temperature of the TB culture medium is 20°C.

[0011] In a preferred embodiment of the application described in this invention, the inducing agent in the TB culture medium is IPTG at a concentration of 0.01-0.05 mM.

[0012] In a preferred embodiment of the application described in this invention, the inducing agent in the TB culture medium is IPTG at a concentration of 0.02 mM.

[0013] In a preferred embodiment of the application described in this invention, the induction rotation speed in the TB culture medium is 120-280 r / min.

[0014] In a preferred embodiment of the application described in this invention, the induction rotation speed in the TB culture medium is 200 r / min.

[0015] In a preferred embodiment of the application described in this invention, the glycosyltransferase UGT91D2 can catalyze the formation of a 1,2-β-D-glycosidic bond from the C13-glycosyl group of stevioside (Rub) to generate steviol glycoside (St), and the enzyme activity is not less than 1309.642 U / L when Rub is used as a substrate.

[0016] In a preferred embodiment of the application described in this invention, the *E. coli* BL21 is a host cell containing the recombinant expression vector pET28a-UGT91D2; the recombinant expression vector pET28a-UGT91D2 is constructed by double digestion of the UGT91D2 gene with restriction endonucleases BamHI and SalI and then ligating it to the pET28a vector.

[0017] As a preferred embodiment of the application described in this invention, the nucleotide sequence of the UGT91D2 gene is shown in SEQ ID NO.1.

[0018] Beneficial effects of this invention:

[0019] This invention novelly proposes an optimized culture medium strategy using TB medium to significantly improve the soluble expression level of recombinant proteins. The presence of glycerol and phosphate buffer in TB medium effectively neutralizes organic acids generated during fermentation, maintaining pH stability during cell growth and resulting in extremely high cell density, thus providing a strong material guarantee for the synthesis of exogenous proteins. This invention innovatively obtains active recombinant proteins using an *E. coli* expression system. By optimizing the culture medium, induction temperature, induction speed, and inducer concentration, the soluble expression level of recombinant proteins is ultimately improved. This invention features a simple operation process and significant effects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0021] Figure 1 This is a plasmid map of pET28a-UGT91D2 in an embodiment of the present invention.

[0022] Figure 2 This is an electrophoresis diagram of the amplification product of the expressed gene UGT91D2 in an embodiment of the present invention.

[0023] Figure 3 This is an SDS-PAGE electrophoresis image of UGT91D2 protein in an embodiment of the present invention; wherein: lane M: protein marker; lane 1: crude UGT91D2 enzyme solution.

[0024] Figure 4 This is the synthetic route for preparing stevioside from stevioside in an embodiment of the present invention.

[0025] Figure 5 These are the HPLC chromatograms of Rub and St standards in embodiments of the present invention. Figure 5 (a) The retention time for the Rub standard is 26.670 min. Figure 5 (b) The retention time of the St standard is 20.998 min.

[0026] Figure 6This is the HPLC chromatogram of the enzyme activity reaction of UGT91D2 with Rub as substrate in an embodiment of the present invention.

[0027] Figure 7 The above are liquid chromatography-mass spectra of product St and St standard reference generated using Rub as substrate in an embodiment of the present invention. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0031] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available. See Table 1 for details.

[0032] Table 1

[0033] name Retail channels Item number Rub Genha B21245-20mg St Genha S59692-5g RA Genha B20694-20mg UDPG Anaiji Chemical E1204970050

[0034] The testing method involved in this invention:

[0035] 1. Quantitative detection of St

[0036] St standard curve determination: St was quantitatively determined using HPLC. A 1.2 mg / mL St standard solution was prepared as a stock solution and gradually diluted to obtain 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL St working solutions. The peak time and peak area were detected using a C18 column at a wavelength of 210 nm. A standard curve was plotted with St concentration as the x-axis and peak area as the y-axis. The equation of the standard curve is y = 3131362x - 17275.

[0037] 2. HPLC conditions

[0038] HPLC analysis was performed using a C18 reverse-phase column (4.6 × 250 mm, 5 μm), at a column temperature of 40 °C. A UV detector with a wavelength of 210 nm was used. The injection volume was 10 μL, the run time for each sample was 45 min, and the mobile phase flow rate was set to 0.8 mL / min. The gradient elution conditions for the mobile phase are shown in Table 2 below.

[0039] Table 2

[0040] time Acetonitrile / % 12.5 mM sodium phosphate buffer / % 0 25 75 13 25 75 15 32 68 40 32 68 42 25 75 45 25 75

[0041] 2. Enzyme activity detection

[0042] The activity of the glycosyltransferase UGT91D2 was determined using Rub as the sugar acceptor (which was identified as the optimal substrate in the initial screening) and UDPG as the sugar donor. The enzyme catalyzes the reaction to produce product St, which can be quantitatively analyzed by UV-HPLC, and the enzyme activity can be calculated accordingly.

[0043] Enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol St per minute under the following reaction conditions: pH=7.4, 30℃, substrate settings: 6 mM UDPG, 9 mM MgCl2 and 3 mM Rub.

[0044] Enzyme activity calculation formula = F × C × 10 6 ×V 总 / St×t×V 酶 The unit is U / mL

[0045] in:

[0046] F: Dilution factor for post-treatment of the reaction solution

[0047] C: The amount of St generated (mg / mL, i.e. g / L) calculated based on the standard curve.

[0048] t: Reaction time (min)

[0049] St: Relative molecular mass is 803 g / mol

[0050] 10 6 1 mol = 10 6 μmol

[0051] V 总 Total volume of the reaction system

[0052] V 酶 : Enzyme liquid volume in the reaction system

[0053] Example 1

[0054] 1. Constructing expression carriers

[0055] The gene sequence of glycosyltransferase UGT91D2 (nucleotide sequence as shown in SEQ ID NO.1) was obtained and given to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, resulting in plasmid pUC57-UGT91D2.

[0056] The upstream and downstream primers UGT91D2-F / UGT91D2-R were designed using software. The UGT91D2 gene was amplified by PCR using the pUC57-UGT91D2 vector as a template. The specific primers are as follows:

[0057] UGT91D2-F: 5′-CACCGGATCCATGGCTACCAGTGACTCATAG- 3′ (SEQ ID NO. 2);

[0058] UGT91D2-R: 5′-CACCGTCGACCTAACTCTATGATCGATGGC-3′ (SEQ ID NO. 3).

[0059] The PCR reaction system consisted of: 25 μL of 2×PCR polymerase PrimeSTAR, 1 μL each of forward and reverse primers, 150 ng of template, and sterile double-distilled water to a final volume of 50 μL. The PCR amplification program was as follows: ① 98℃ for 3 min; ② 98℃ for 10 s, 65℃ for 5 s, and 72℃ for 85 s, for a total of 33 cycles; ③ 72℃ extension for 10 min. After the reaction, the product size was determined by 1% agarose gel electrophoresis.

[0060] The UGT91D2 gene, obtained by double digestion with restriction endonucleases BamHI and SalI, was amplified and then coupled with the pET28a vector. After nucleic acid gel recovery, linear plasmid fragments were obtained. The UGT91D2 target gene was ligated into the pET28a vector, and the ligation product was transformed into E. coli Top10 competent cells. Transformants were selected and sequenced to obtain the correct recombinant plasmid pET28a-UGT91D2.

[0061] 2. Construction of recombinant strains

[0062] Take 2-4 µL of recombinant plasmid pET28a-UGT91D2 and add it to BL21 (DE3). Let it stand in crushed ice for 30 min. Then, place the mixture in a 42℃ water bath for 90 s heat shock. Quickly transfer it to crushed ice to cool for 3 min. Add 900 µL of LB medium and revive at 200 rpm and 37℃ for 1 h. Spread it on LB solid plates containing kan (100 µg / ml) resistance and incubate it upside down at 37℃ for 12 h. Transformants that grow normally are the recombinant strains. Preserve the strains according to the preservation system and store them at -80℃.

[0063] 3. Shake-flask fermentation

[0064] Seed culture preparation: A single colony of the above recombinant strain was inoculated into 5 ml of LB liquid medium and cultured at 37°C and 200 rpm for 12 h. Induction expression stage: The seed culture was inoculated into 150 ml of LB medium at a 1% inoculation rate. Cultured at 37°C with shaking at 200 rpm until OD... 600nm The concentration of the culture medium was 0.6-0.8. IPTG was added to a final concentration of 0.05 mM. The culture medium temperature was lowered to 16℃, and induction culture was continued at 200 rpm for 24 h. The bacterial suspension was centrifuged at 10000 rpm for 10 min at 4℃, the culture medium was removed, and the cells were washed twice with 15 mL PBS buffer. 1 g of wet bacteria was resuspended in 10 mL PBS buffer, and the cells were sonicated on ice. The sonication conditions were: power 144 W, sonication for 2 s, pause for 3 s, time 25 min. After disruption, the cells were centrifuged at 10000 rpm for 10 min at 4℃, and the supernatant was collected. The supernatant is the crude enzyme solution.

[0065] 4. SDS-PAGE detection of protein expression

[0066] The raw crude enzyme solution of UGT91D2 was subjected to SDS-PAGE. The specific steps are as follows:

[0067] (1) Gel preparation: Using the polyacrylamide gel preparation kit from Novizan, a 1.0 mm thick gel was prepared according to the kit instructions. The lower and upper gels were mixed separately and injected into the gaps of the gel preparation plate. A comb was inserted and waited for 15 min for it to solidify.

[0068] (2) Preparation of electrophoresis buffer: Weigh 21.6 g of glycine, 4.5 g of tris(hydroxymethyl)aminomethane (Tris), and 1.5 g of sodium dodecyl sulfate (SDS) and dissolve them in 1.5 L of deionized water;

[0069] (3) Sample preparation: Mix the electrophoresis loading buffer with the crude enzyme solution or the precipitate resuspension solution and boil in a water bath for 10 min;

[0070] (4) Sample loading and electrophoresis: Add 10 µL of sample to the sample well, run at 80 V until the sample passes through the pink stacking gel, then adjust the voltage to 120 V until the end;

[0071] (5) Coomassie Brilliant Blue staining and destaining: The gel was stained in Coomassie Brilliant Blue staining solution for 40 min. After staining, it was placed in destaining solution for 2 h, and then replaced with fresh destaining solution overnight. Finally, the gel was photographed and recorded using a gel imaging system.

[0072] 5. Enzyme activity assay

[0073] The total reaction volume was 600 µL, containing 400 µL of crude enzyme solution and 200 µL of reaction solution. The reaction solution contained 6 mM UDPG, 9 mM MgCl2, and 3 mM (Rub, St, RA). The reaction was carried out at 30 °C for 12 h, and then 1.8 mL of acetonitrile was added to terminate the reaction. The reaction mixture was filtered through a 0.22 µm organic filter membrane and then packaged into a liquid chromatography sample vial. The products St, RE, and RD can be quantitatively detected by liquid chromatography.

[0074] Figure 1 The pET28a-UGT91D2 plasmid map; Figure 2 Using pUC57-UGT91D2 as template DNA, the target gene was amplified from the synthesized plasmid pUC57-UGT91D2 by PCR using primers UGT91D2-F and UGT91D2-R. The agarose gel electrophoresis results are as follows: Figure 2 As shown, the main band of the PCR product is approximately 1.5 kbp in size, which is consistent with the expected length. Figure 3 The original supernatant of the UGT91D2 recombinant strain after cell disruption was analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 3 As shown, the recombinant strain expressed protein at approximately 53 kDa, and the size of the target protein was consistent with expectations. Figure 4 Synthetic route for preparing steviol glycosides from steviosides Figure 5 The HPLC chromatograms are for Rub and St standards. Figure 5 (a) The retention time for the Rub standard is 26.670 min. Figure 5 (b) The retention time of the St standard is 20.998 min. Figure 6 The HPLC chromatogram shows the reaction of Rub as substrate with the original crude enzyme solution of UGT91D2. The results show that the peak time of Rub is the same as that of Rub standard at 26.435 min. At the same time, a peak appears at 20.925 min, which is consistent with the peak time of standard St. It is preliminarily identified as St. Figure 7 The substances that elute at 20.925 min in the St standard control and the reaction solution were simultaneously analyzed by liquid chromatography-mass spectrometry (LC-MS). The results showed that the molecular weight of the St standard control was 803 in the LC-MS spectrum, and the molecular weight of the substance in the reaction solution was also 803, thus confirming the formation of product St.

[0075] Quantitative analysis of the product was performed by HPLC. UGT91D2 can generate St using Rub as a substrate. The purified product was verified. When Rub (molecular weight 642) was used as a substrate, the molecular weight of the product was 803, which was consistent with the molecular weight of St. Therefore, the product was confirmed to be St. However, the products generated using St and RA as substrates were almost undetectable. Therefore, Rub was determined to be the optimal substrate and used for subsequent enzyme activity assays.

[0076] Example 2

[0077] Effects of different culture media on enzyme production during UGT91D2 fermentation

[0078] The only difference from Example 1 is that the culture medium type in the induction expression stage of step 3 is adjusted, and different fermentation media are set: TB (Example 2-1), LB-sorbitol (Example 2-2), and 2×YT (Example 2-3).

[0079] Example 3

[0080] Effect of different induction temperatures on enzyme production during UGT91D2 fermentation

[0081] Based on the culture medium optimization in Example 2, the induction temperature was further optimized. The difference from Example 1 is that...

[0082] In step 3, the TB medium determined in Example 2-1 was used for the induction expression stage, and different induction temperatures were set: 20℃ (Example 3-1), 25℃ (Example 3-2), 30℃ (Example 3-3), and 37℃ (Example 3-4).

[0083] Example 4

[0084] Effects of different inducer concentrations on enzyme production during UGT91D2 fermentation

[0085] Based on the optimization of the preceding culture medium and induction temperature, the concentration of the inducer was further optimized. Compared with Example 1, the difference is that the TB culture medium determined in Example 2-1 was used in the induction expression stage of step 3, and the induction temperature of 20°C determined in Example 3-1 was used. At the same time, different inducer concentrations were set for testing, specifically: 0.02 mM (Example 4-1), 0.1 mM (Example 4-2), 0.2 mM (Example 4-3), and 0.4 mM (Example 4-4).

[0086] Example 5

[0087] Effect of different induction speeds on enzyme production during UGT91D2 fermentation

[0088] Based on the optimization of the preceding culture medium, induction temperature, and inducer concentration, the induction speed was further optimized. Compared with Example 1, the difference is that in step 3, the TB culture medium determined in Example 2-1, the induction temperature of 20℃ determined in Example 3-1, and the inducer concentration of 0.02 mM determined in Example 4-1 were used for the induction expression stage. At the same time, different induction speeds were set for testing, specifically: 120 rpm (Example 5-1), 160 rpm (Example 5-2), 240 rpm (Example 5-3), and 280 rpm (Example 5-4).

[0089] Comparative Example 1

[0090] The only difference from Example 1 is that an E. coli strain without the UGT91D2 target gene was used; all other experimental conditions were exactly the same as in Example 1.

[0091] Comparative Example 2

[0092] The only difference from Example 1 is that an Escherichia coli strain containing the UGT91D2 target gene was used, and no inducer was added during the induction phase; all other conditions were the same as in Example 1.

[0093] Example 6

[0094] Results Measurement

[0095] 1. The effects of different culture media on enzyme activity are shown in Table 3.

[0096] Table 3. Enzyme activities under different culture media

[0097] Example Enzyme activity (U / L) Example 1 (LB) 785.751 Example 2-1 (TB) 1069.585 Example 2-2 (LB-sorbitol) 576.674 Examples 2-3 (2×YT) 182.754

[0098] Table 3 shows that the composition of the culture medium has a significant impact on enzyme activity, with the highest and lowest enzyme activities differing by nearly six times. Specifically, TB medium induced a 36% increase in enzyme activity compared to LB medium. This is attributed to the presence of phosphate buffer and glycerol in TB medium, which provides a more stable environment and more sustained nutrition for cell growth and protein expression. Conversely, the addition of small molecules such as sorbitol to LB medium resulted in a significant decrease in enzyme activity, possibly because many bacteria utilize sorbitol much less efficiently than glycerol or glucose. Although sorbitol can increase the osmotic pressure of the medium, it may exceed the optimal range for bacterial growth, thus inhibiting it. This experiment clearly demonstrates that even small changes in the composition of the culture medium can have a significant impact on the final enzyme activity, making medium optimization a crucial step in increasing enzyme yield.

[0099] 2. The effect of different induction temperatures on enzyme activity is shown in Table 4.

[0100] Table 4. Enzyme activity at different induction temperatures

[0101] Example Enzyme activity (U / L) Example 2-1 (16℃) 1069.585 Example 3-1 (20℃) 1217.104 Example 3-2 (25℃) 223.727 Example 3-3 (30℃) 181.754 Examples 3-4 (37℃) 171.876

[0102] Table 4 shows that the highest enzyme activity was obtained under induction at 20℃. When the temperature increased to 25℃, the enzyme activity dropped sharply, by more than 80%. At 30℃ and 37℃, the enzyme activity was at its lowest level, with no significant difference between the two. These results indicate that lower induction temperatures greatly promote the soluble expression and correct folding of the target protein, while higher temperatures cause excessively rapid protein synthesis, leading to inclusion body formation and the formation of inactive proteins. Therefore, 20℃ was determined to be the optimal temperature for protein expression.

[0103] 3. The effect of different inducer concentrations on enzyme activity is shown in Table 5.

[0104] Table 5. Enzyme activity at different inducer concentrations

[0105] Example Enzyme activity (U / L) Example 3-1 (0.05 mM) 1217.104 Example 4-1 (0.02 mM) 1309.642 Example 4-2 (0.1 mM) 929.681 Example 4-3 (0.2 mM) 439.157 Example 4-4 (0.4 mM) 299.507

[0106] Table 5 shows that the concentration of the inducer IPTG significantly regulates the activity of the recombinant protease. The results indicate that enzyme activity gradually decreases with increasing IPTG concentration. This suggests that lower concentrations of the inducer are sufficient to effectively initiate the expression of the exogenous protein, while excessively high IPTG concentrations exacerbate the bacterial metabolic burden, inhibit cell growth, and lead to protein misfolding and inclusion body formation, thus significantly reducing the yield of active enzymes. This experiment determined 0.02 mM as the optimal induction concentration.

[0107] 4. The effect of different rotation speeds on enzyme activity is shown in Table 6.

[0108] Table 6. Enzyme activity at different rotation speeds

[0109] Example Enzyme activity (U / L) Example 5-1 (120 r / min) 967.02 Example 5-2 (160 r / min) 1221.016 Example 4-1 (200 r / min) 1309.642 Example 5-3 (240 r / min) 972.135 Example 5-4 (280 r / min) 878.080

[0110] When the rotation speed increased from 120 r / min to 200 r / min, the enzyme activity significantly increased from 967.02 to 1309.64. Within this range, increasing the rotation speed had a positive promoting effect on enzyme activity. However, when the rotation speed exceeded 200 r / min, further increasing the rotation speed caused the enzyme activity to drop sharply from the peak of 1309.64 to 972.14. This is likely because excessively high rotation speeds generate strong fluid shear forces, leading to denaturation of soluble proteins. Therefore, rotation speed has a very significant impact on enzyme activity, and this experiment determined that 200 r / min is the optimal rotation speed for maximizing enzyme activity.

[0111] 5. The effects of Example 1 and Comparative Examples 1-2 on enzyme activity are shown in Table 7.

[0112] Table 7 Effects of Example 1 and Comparative Examples 1-2 on enzyme activity

[0113] Example Enzyme activity (U / L) Example 1 (LB) 785.751 Comparative Example 1 — Comparative Example 2 —

[0114] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

[0115] The gene sequence of UGT91D2 is SEQ ID NO.1:

[0116]

Claims

1. The application of TB culture medium in the soluble expression level of glycosyltransferase UGT91D2, characterized by: The TB medium maintains pH stability and increases cell density during cell growth in the E. coli BL21 expression system, thereby enhancing the soluble expression level of recombinant UGT91D2 protein.

2. The application according to claim 1, characterized in that: The culture temperature of the TB medium is 16-25℃.

3. The application according to claim 2, characterized in that: The culture temperature of the TB medium is 20℃.

4. The application according to claim 1, characterized in that: The inducer in the TB culture medium is IPTG at a concentration of 0.01-0.05 mM.

5. The application according to claim 4, characterized in that: The inducer in the TB culture medium is IPTG at a concentration of 0.02 mM.

6. The application according to claim 1, characterized in that: The induction rotation speed in the TB medium was 120-280 r / min.

7. The application according to claim 6, characterized in that: The induction rotation speed in the TB medium was 200 r / min.

8. The application according to claim 1, characterized in that: The glycosyltransferase UGT91D2 can catalyze the formation of a 1,2-β-D-glycosidic bond from the C13-glycosyl group of stevioside to generate stevioside, and its enzyme activity is not less than 1309.642 U / L when Rub is used as a substrate.

9. The application according to claim 1, characterized in that: The *Escherichia coli* BL21 is a host cell containing the recombinant expression vector pET28a-UGT91D2; the recombinant expression vector pET28a-UGT91D2 is constructed by double digestion of the UGT91D2 gene with restriction endonucleases BamHI and SalI and then ligating it into the pET28a vector.

10. The application according to claim 1, characterized in that: The nucleotide sequence of the UGT91D2 gene is shown in SEQ ID NO.1.

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