A glycosyltransferase mutant and its encoding gene and use of catalytic Reb D

Abstract

The present invention provides a glycosyltransferase mutant and a coding gene and a use of catalyzing Reb D, belonging to the field of bioenzyme technology. The present invention provides a glycosyltransferase mutant capable of efficiently catalyzing the biosynthesis of Reb D from Reb A, wherein the glycosyltransferase mutant is subjected to single-point and multi-point mutations at positions 147 (glutamine Q), 206 (glutamic acid E), 209 (phenylalanine F) and 268 (glycosylamine G) of the amino acid sequence of PmUGT1, and the catalytic activity can be increased by up to 9.27 times compared with the wild-type PmUGT1.

Description

Technical Field

[0001] The invention belongs to the technical field of biological enzymes, and particularly relates to a glycosyltransferase mutant and a coding gene and uses of catalytic Reb D. Background Art

[0002] Stevioside (Stevia), also known as stevia sugar, is a new type of sweetener extracted from the leaves of the plant Stevia. Since stevioside does not produce calories, does not increase blood sugar levels, and is 300 times sweeter than sucrose, it has been used as a non-nutritive sweetener in sweet foods in some countries for hundreds of years. Rebaudioside A (RebA) is the main steviol glycoside, which is 300 times sweeter than sucrose. However, the bitter taste of RebA affects the taste, and its price is slightly lower than Rebaudioside D (RebD).

[0003] However, since the content of RebD in stevia is very low, the cost of plant extraction and purification is huge, and the output is far from meeting market demand. Therefore, RebD cannot be produced by direct extraction from plants, and can only be synthesized artificially. Artificial synthesis of RebD requires glycosylation reaction, and there are two ways: chemical synthesis and enzymatic synthesis. Chemical glycosylation usually requires cumbersome protection and deprotection steps, and has low specificity and yield. Enzymatic glycosylation shows high efficiency, stereo and regional specificity, can directly and quickly produce glycosidic bonds, and is environmentally friendly. So far, many glycosyltransferases from different sources have been identified to have the activity of catalyzing the synthesis of RebD, but these glycosyltransferases generally have low activity and cannot tolerate high substrate concentrations. In addition, some enzymes have side reactions, which will produce homologues of RebD, increasing the pressure of downstream purification. Summary of the invention

[0004] The present invention provides a glycosyltransferase mutant and a coding gene and use of the catalytic Reb D. The glycosyltransferase mutant can utilize low-value Reb A to efficiently catalyze the synthesis of high-value Reb D.

[0005] The present invention provides a glycosyltransferase mutant, comprising single-point or multi-point mutation of the amino acid sequence of glycosyltransferase UGT1;

[0006] The amino acid sequence of the glycosyltransferase UGT1 is shown in SEQ ID No.1.

[0007] In a specific embodiment of the present invention, single-point or multi-point mutations are performed on positions 147, 206, 209 and 268 of the amino acid sequence shown in SEQ ID No. 1.

[0008] In a specific embodiment of the present invention, the single-point or multi-point mutation includes any one or more of the following combinations: mutating the 147th glutamine of the amino acid sequence shown in SEQ ID No.1 to isoleucine, mutating the 206th glutamic acid to arginine, mutating the 209th phenylalanine to proline and mutating the 268th glycine to methionine.

[0009] The present invention also provides a gene encoding the glycosyltransferase mutant.

[0010] The invention also provides a primer pair for amplifying the gene.

[0011] The present invention also provides a recombinant vector comprising the above gene and expressing the above glycosyltransferase mutant.

[0012] The present invention also provides a recombinant cell comprising the recombinant vector and expressing the glycosyltransferase mutant.

[0013] The present invention also provides the use of the above-mentioned glycosyltransferase mutant or the recombinant glycosyltransferase mutant produced by the above-mentioned recombinant cell in catalyzing the biosynthesis of Reb D from Reb A.

[0014] The present invention also provides a method for catalyzing the biosynthesis of Reb D from Reb A, comprising using Reb A as a substrate, UDPG as a glucose donor, and utilizing the above-mentioned glycosyltransferase mutant or the recombinant glycosyltransferase mutant produced by the above-mentioned recombinant cell to carry out an enzymatic reaction to biosynthesize Reb D.

[0015] In a specific embodiment of the present invention, the temperature of the enzymatic reaction is 40° C. and the time is 12 h.

[0016] Beneficial effect: The present invention has discovered a glycosyltransferase PmUGT1 (WP_192997595.1) with the activity of catalyzing the synthesis of rebaudioside D from rebaudioside A, and the glycosyltransferase PmUGT1 is derived from bacteria Priestia megaterium Compared with plant-derived glycosyltransferases, such as EUGT11 and UGTSL2, it can be functionally expressed in Escherichia coli BL21 (DE3), has excellent solubility, and can catalyze the biosynthesis of Reb A to Reb D, while having high regioselectivity. Therefore, the present invention provides a glycosyltransferase mutant that can efficiently catalyze the biosynthesis of Reb A to Reb D, wherein the glycosyltransferase mutant is a single-point and multi-point mutation at position 147 (glutamine Q), position 206 (glutamic acid E), position 209 (phenylalanine F), and position 268 (glycosylamine G) of the amino acid sequence of PmUGT1, and the catalytic activity can be increased by up to 9.27 times compared with the wild-type UGT1. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the plasmid map of the recombinant plasmid pET24a-PmUGT;

[0018] Figure 2 The electrophoresis diagram of plasmid enzyme digestion fragments;

[0019] Figure 3 This is the liquid phase diagram of rebaudioside D (RD) standard;

[0020] Figure 4 This is the liquid spectrum of the conversion fluid. DETAILED DESCRIPTION

[0021] The present invention provides a glycosyltransferase mutant, comprising single-point or multi-point mutation of the amino acid sequence of glycosyltransferase UGT1;

[0022] The amino acid sequence of the glycosyltransferase UGT1 is shown in SEQ ID No. 1: MGNVLVIGFPGEGHVNPSLGIVKELMAQGEKVVYYGIEDYAEKIRKSGAEFREYEDFRPSLDMSNRMTKEESYDRYEIMNLFLNESENIITKIMSETKHETYDYVLYDYHLLAGSAVADLLQLPKVSLCTTFALNKELATCMMPSGQTEPDQSSFYPQFEKSLNKFNNQYNTELKDSMDIMSNPGNITLVFTSEFFQ PNVSEFTSEYKFIGPSIVKRLDVEDPSYLQNENEKIIFVSMGTIFNQQLEIYNLCVEALKDFEGKVILSIGKNTKTEELAHIPENFVVKQYVPQLEILKRAD LFITHGGMNSSSEGLYYDTPLVVIPMAVDQFMVGDRVQKLGAGVKLDKNNVSAELIKNTVNDVLTTHTYKQHAQQIGESLRSAGGYKQGVKEIFKMVPVKA.

[0023] In a specific embodiment of the present invention, single-point or multi-point mutations are performed on positions 147, 206, 209 and 268 of the amino acid sequence shown in SEQ ID No. 1. In a specific embodiment of the present invention, the single-point or multi-point mutations include any one or more of the following combinations: mutating glutamine at position 147 of the amino acid sequence shown in SEQ ID No. 1 to isoleucine (Q147I), mutating glutamic acid at position 206 to arginine (E206P), mutating phenylalanine at position 209 to proline (F209R) and mutating glycosylamine at position 268 to methionine (G268M). After single-point mutation, the enzyme activity of mutant Q147I increased by 1.28 times compared with the wild type, the enzyme activity of E206P increased by 5.27 times compared with the wild type, the enzyme activity of F209R increased by 0.89 times compared with the wild type, and the enzyme activity of G268M increased by 2.37 times compared with the wild type; after double-point mutation, the enzyme activity of mutant Q147I / E206P increased by 3.12 times compared with the wild type, and the enzyme activity of mutant Q147I / F Compared with the wild type, the enzyme activity of 209R increased by 2.24 times, and the enzyme activity of mutant Q147I / G268M increased by 1.98 times, which was 2.84 U / mg. After three-point mutations, the enzyme activity of mutant Q147I / F209R / E206P increased by 5.41 times, and the enzyme activity of mutant Q147I / F209R / G268M increased by 3.76 times, which was 4.54 U / mg. After four-point mutations, the enzyme activity of mutant Q147I / F209R / E206P / G268M increased by 9.27 times, which was 9.8 U / mg.

[0024] The present invention also provides a gene encoding the glycosyltransferase mutant.

[0025] The coding gene sequence of the glycosyltransferase UGT1 described in the present invention can be WP_192997595.1, or it can be a homologous gene of the WP_192997595.1, and any homologous gene with a homology exceeding 85% and having the same function as UGT1 can be used.

[0026] The invention also provides a primer pair for amplifying the gene.

[0027] In a specific embodiment of the present invention, the PmUGT gene (SEQ ID No. 2) shown in WP_192997595.1 is connected to pET24a EcoR I- Hind III site, forming Figure 1The recombinant plasmid pET24a-PmUGT shown; using the recombinant plasmid pET24a-PmUGT as a template, single point mutations Q147I, E206P, F209R and G268M can be constructed according to site-directed mutagenesis primers, wherein the primers used can be as shown below, thereby obtaining recombinant vectors pET24a-PmUGT (Q147I), pET24a-PmUGT (E206P), pET24a-PmUGT (F209R) and pET24a-PmUGT (G268M).

[0028] SEQ ID No.2:

[0029]

[0030] In one embodiment of the present invention, the above mutation sites are also subjected to combined mutation, and whole plasmid PCR is performed using the recombinant plasmid pET24a-PmUGT (Q147I) as a template to obtain PmUGT (Q147I / E206P), PmUGT (Q147I / F209R), and PmUGT (Q147I / G268M) mutants; the recombinant plasmid pET24a-PmUGT (Q147I / F20 9R) was used as a template for whole-plasmid PCR to obtain PmUGT (Q147I / F209R / E206P) and PmUGT (Q147I / F209R / G268M) mutants; whole-plasmid PCR was performed using the recombinant plasmid pET24a-PmUGT (Q147I / F209R / E206P) as a template to obtain PmUGT (Q147I / F209R / E206P / G268M).

[0031] Q147I site-directed mutagenesis primers:

[0032] F1 (SEQ ID No. 3): tgccaagtggtataacagaacctATGATGCCAAGTGGTATAACAGAACC;

[0033] R1 (SEQ ID No. 4): ctgttataccacttggcatcataAGGTTCTGTTATACCACTTGGCATC;

[0034] E206P site-directed mutagenesis primers:

[0035] F1 (SEQ ID No.5): ctgaatttacgagcccgtacaGTATCTGAATTTACGAGCCCGTACA;

[0036] R1 (SEQ ID No. 6): acgggctcgtaaattcagataTGTACGGGCTCGTAAATTCAGA;

[0037] F209R site-directed mutagenesis primers:

[0038] F1 (SEQ ID No.7): acaagcgcattggcctCAAGCGCATTGGCCCTTC;

[0039] R1 (SEQ ID No. 8): aggggccaatgcgcttgtGCCAATGCGCTTGTACGG;

[0040] G268M site-directed mutagenesis primers:

[0041] F1 (SEQ ID No. 9): atctattatgaaaaatacaaCTATTATGAAAAATACAAAAACTGAAGAATTA;

[0042] R1 (SEQ ID No. 10): gtatttttcataatagataaTTTTTCATAATAGATAAAATAACTTTACCCTC.

[0043] The present invention also provides a recombinant vector comprising the above gene and expressing the above glycosyltransferase mutant.

[0044] The recombinant vector of the present invention can use various types of vectors as backbone vectors, such as pET series, pKK223-3, pGEX series, pMAL series, etc. In one embodiment, pET24a (+) is used as the backbone vector to construct a recombinant vector carrying single point mutations and multiple point mutations.

[0045] The present invention also provides a recombinant cell comprising the recombinant vector and expressing the glycosyltransferase mutant.

[0046] The recombinant cells of the present invention are based on Escherichia coli, Bacillus subtilis, yeast, etc. In the embodiments, Escherichia coli BL21 (DE3) is used as an example to illustrate that the above recombinant vector is transformed into the Escherichia coli to construct a recombinant Escherichia coli cell.

[0047] The present invention also provides the use of the above-mentioned glycosyltransferase mutant or the recombinant glycosyltransferase mutant produced by the above-mentioned recombinant cell in catalyzing the biosynthesis of Reb D from Reb A.

[0048] The glycosyltransferase mutant of the present invention has the function of efficiently catalyzing the biosynthesis of Reb D from Reb A.

[0049] The present invention also provides a method for catalyzing the biosynthesis of Reb D from Reb A, comprising using Reb A as a substrate, UDPG as a glucose donor, and utilizing the above-mentioned glycosyltransferase mutant or the recombinant glycosyltransferase mutant produced by the above-mentioned recombinant cell to carry out an enzymatic reaction to biosynthesize Reb D.

[0050] In one embodiment of the present invention, the enzymatic reaction system includes substrate, enzyme, reaction pH, reaction temperature, reaction time, etc. In one embodiment of the present invention, the enzymatic reaction temperature is 40° C., pH 7.5, and the reaction time is 12 h.

[0051] In order to further illustrate the present invention, a glycosyltransferase mutant and encoding gene provided by the present invention and the use of catalytic Reb D are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0052] The invention embodiment used Figure 1 The construction method of the recombinant plasmid pET24a-PmUGT shown in the figure is as follows: General Biotechnology (Anhui) Co., Ltd. was commissioned to perform full gene synthesis. EcoR I-- Hind III is the restriction site, and the restriction fragments are recovered and connected to the pET24a vector that is also digested and recovered.

[0053] The definition of enzyme activity in the embodiments of the present invention is: the amount of enzyme required to produce 1 μM Reb D per minute at pH 7.5 and 40°C.

[0054] Example 1 Directed evolution of glycosyltransferase PmUGT

[0055] (1) Using the recombinant plasmid pET24a-PmUGT as a template, design EcoR I restriction site upstream primer PmUGT-F and Hind III restriction site downstream primer PmUGT-R. Add a certain concentration of Mg to the ordinary PCR reaction system. 2+ and Mn 2+ Perform error-prone PCR;

[0056] PmUGT-F (SEQ ID No. 11): GAATTCATGGATGGGAAATGTATTAGT;

[0057] PmUGT-R (SEQ ID No. 12): CCACTCGAGTTACGCTTTAATTCGAA;

[0058] (2) Error-prone PCR reaction system (50 μL): 10×Easy Taq® Buffer 5 μL, dNTPs (2.50 mmol / L) 4 μL, template 10 ng, forward and reverse primers (10 μmol / L) 1 μL, Easy Taq® DNA Polymerase 0.50 μL, and different concentrations of Mg 2+ (final concentrations 2, 4, 6, 8 and 10 mmol / L) and Mn 2+ (final concentrations 0, 0.10, 0.30, 0.50, 0.70 and 0.90 mmol / L), and finally sterile ddH 2 O replenishment system, determine the optimal Mg based on the mutation rate2+ and Mn 2+ concentrations were used in subsequent experiments.

[0059] (3) PCR amplification conditions: hot start at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 3.5 min, 35 cycles; extension at 72°C for another 5 min; storage at 4°C.

[0060] (4) Perform gel electrophoresis on the error-prone PCR reaction system and use a DNA gel recovery kit to recover the target DNA fragment. EcoR I and Hind III, double-digest the target DNA fragment and pET24a expression vector at 37°C for 4 h. The double-digested bands were verified by electrophoresis and recovered, and the target DNA bands were connected to the vector fragments at 16°C overnight using T4 DNA ligase. The ligation products were transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, transferred to LB plates containing 50 μg / mL kanamycin, and cultured at 37°C overnight to obtain a mutant library of about 1000 transformants.

[0061] (5) Place the single colony picked for transformation in a 96-well plate and culture it. Add 0.5 mM IPTG and induce the culture at 25°C for 16 h. Collect the bacteria by centrifugation and resuspend them in 200 μL buffer. Add 10 mg / ml lysozyme and centrifuge to obtain the supernatant, which is the crude enzyme solution.

[0062] (6) Enzyme activity test system: Add 2 mM rebaudioside A, 5 mM UDPG and 0.1 mL crude enzyme solution to a 200 μL reaction system. The reaction pH was 7.5 and the reaction temperature was 40°C. The reaction was carried out for 2 h. The reaction was inactivated by boiling in water for 3 min. The supernatant was collected by centrifugation and the content of rebaudioside D was detected by HPLC.

[0063] (7) The mutants screened were sequenced and the mutation sites were determined to be 147 (glutamine Q), 206 (glutamate E), 209 (phenylalanine F), and 268 (glycine G). The activity of the glycosyltransferase PmUGT mutant Q147I was 1.28 times higher than that of the wild type, the activity of E206P was 5.27 times higher than that of the wild type, the activity of F209R was 0.89 times higher than that of the wild type, and the activity of G268M was 2.37 times higher than that of the wild type. One unit of enzyme activity U is the amount of enzyme required to catalyze the production of 1 micromole of product from the substrate within 1 minute under specific conditions. Specifically, 2 mM rebaudioside A, 5 mM UDPG and 100 μL crude enzyme solution were added to a 200 μL reaction system, the reaction pH was 7.5, the reaction temperature was 40°C, after 2 h of reaction, the product was boiled and inactivated, the supernatant was collected by centrifugation, the content of rebaudioside D was detected by HPLC, and then the enzyme activity was calculated.

[0064] (8) Design of site-directed mutagenesis primers

[0065] The Q147I site-directed mutagenesis primers are SEQ ID No.3 and SEQ ID No.4; the E206P site-directed mutagenesis primers are SEQ ID No.5 and SEQ ID No.6; the F209R site-directed mutagenesis primers are SEQ ID No.7 and SEQ ID No.8; and the G268M site-directed mutagenesis primers are SEQ ID No.9 and SEQ ID No.10.

[0066] (9) The mutation sites of (7) were combined for mutation. The recombinant plasmid pET24a-PmUGT (Q147I) was used as a template and SEQ ID No. 5 and SEQ ID No. 6 were used as primers to perform whole plasmid PCR to obtain PmUGT (Q147I / E206P). The recombinant plasmid pET24a-PmUGT (Q147I) was used as a template and SEQ ID No. 7 and SEQ ID No. 8 were used as primers to perform whole plasmid PCR to obtain PmUGT (Q147I / F209R). The recombinant plasmid pET24a-PmUGT (Q147I) was used as a template and SEQ ID No. 9 and SEQ ID No. 10 were used as primers to perform whole plasmid PCR to obtain PmUGT (Q147I / G268M) mutant.

[0067] (10) Using the recombinant plasmid pET24a-PmUGT (Q147I / F209R) as a template and SEQ ID No. 5 and SEQ ID No. 6 as primers, whole plasmid PCR was performed to obtain PmUGT (Q147I / F209R / E206P); using the recombinant plasmid pET24a-PmUGT (Q147I / F209R) as a template and SEQ ID No. 9 and SEQ ID No. 10 as primers, whole plasmid PCR was performed to obtain the PmUGT (Q147I / F209R / G268M) mutant;

[0068] (11) Using the recombinant plasmid pET24a-PmUGT (Q147I / F209R / E206P) as a template and SEQ ID No. 9 and SEQ ID No. 10 as primers, whole plasmid PCR was performed to obtain PmUGT (Q147I / F209R / E206P / G268M);

[0069] The electrophoresis of plasmid digestion fragments is shown in Figure 2 As shown, the enzyme activity of PmUGT (Q147I / F209R / E206P / G268M) was increased by 9.27 times compared with the wild type.

[0070] (12) The recombinant bacteria containing two recombinant plasmids were induced to express according to the above method;

[0071] (13) In the best case, 100 mM rebaudioside A, 8 mM UDP, 800 mM sucrose, and 5 mM MgCl were added to the 100 ml reaction system. 2 and 50 mL crude enzyme solution, reaction pH 7.5, reaction temperature 40 ℃, after reaction for 12 h, boil in boiling water for 3 min to inactivate, centrifuge and take supernatant, HPLC detection of residual content of rebaudioside A is 9.5 mM; add 1 L pure water, heat to above 95 ℃ to dissolve Reb D, and then detect the content of Reb D by HPLC, converted to 100 ml system, the concentration of Reb D is 100.8 g / L.

[0072] Comparative Example 1

[0073] (1) Recombinant Escherichia coli BL21 (DE3) expressing glycosyltransferase PmUGT (WP_192997595.1) was constructed. The expression vector was pET24a (+). The amino acid sequence of glycosyltransferase PmUGT is as follows:

[0074] MGNVLVIGFPGEGHVNPSLGIVKELMAQGEKVVYYGIEDYAEKIRKSGAEFREYEDFRPSLDMSNRMTKEESYDRYEIMNLFLNESENIITKIMSETKHETYDYVLYDYHLLAGSAVADLLQLPKVSLCTTFALNKELATCMMPSGQTEPDQSSFYPQFEKSLNKFNNQYNTELKDSMDIMSNPGNITLVFTSEFFQPNV SEFTSEYKFIGPSIVKRLDVEDPSYLQNENEKIIFVSMGTIFNQQLEIYNLCVEALKDFEGKVILSIGKNTKTEELAHIPENFVVKQYVPQLEILKRADL FITHGGMNSSSEGLYYDTPLVVIPMAVDQFMVGDRVQKLGAGVKLDKNNVSAELIKNTVNDVLTTHTYKQHAQQIGESLRSAGGYKQGVKEIFKMVPVKA;

[0075] (2) The recombinant bacteria were activated and cultured overnight in LB liquid medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl) containing 50 μg / ml kanamycin at 37°C as seed solution;

[0076] (3) Inoculate 2% of the seed solution into 2×YT medium (16 g / L peptone, 10 g / L yeast powder, 5 g / L NaCl) containing 50 μg / ml kanamycin, add 0.5 mM IPTG and induce culture at 25°C for 16 h, collect the cells by centrifugation, resuspend in buffer and dilute to 10 times, disrupt by ultrasonication and centrifuge to obtain the supernatant as the crude enzyme solution;

[0077] (3) Add 2 mM rebaudioside A, 5 mM UDPG and 100 μL crude enzyme solution to a 200 μL reaction system. The reaction pH was 7.5 and the reaction temperature was 40°C. After 2 h of reaction, the mixture was inactivated by boiling and centrifuged to obtain the supernatant. The content of rebaudioside D was detected by HPLC.

[0078] (4) An Agilent ZORBAX SB-C18 column (4.6×250 mm 5 μm) was used with an injection volume of 5 μL, a column temperature of 40°C, mobile phase A consisting of acetonitrile and mobile phase B consisting of 0.2% formic acid in water, a flow rate of 0.5 ml / min, and analysis at 210 nm.

[0079] The specific procedures are shown in Table 1.

[0080] Table 1 Chromatographic procedures

[0081]

[0082] (5) Enzyme activity definition: The amount of enzyme required to produce 1 μM Reb D per minute under the conditions of reaction pH 7.5 and temperature 40°C. The liquid phase diagram of RD standard is as follows: Figure 3 As shown;

[0083] (6) Optimal example: 2 mM Reb A, 5 mM UDPG and 1 ml crude enzyme solution, reaction pH 7.5, reaction temperature 40°C, reaction time 2 h, the liquid spectrum of the conversion solution is as follows: Figure 4 As shown, the unit enzyme activity is 9.8 U / mg.

[0084] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A glycosyltransferase mutant, characterized in that: The glycosyltransferase mutant is obtained by performing single-point or multi-point mutation on the amino acid sequence of glycosyltransferase UGT1; The amino acid sequence of the glycosyltransferase UGT1 is shown in SEQ ID No. 1; The single-point or multi-point mutation is the following single-point or multi-point mutation of the amino acid sequence shown in SEQ ID No.1: Q147I, E206P, F209R, G268M, Q147I / E206P, Q147I / F209R, Q147I / G268M, Q147I / F209R / E206P, Q147I / F209R / G268M or Q147I / F209R / E206P / G268M.

2. A gene encoding the glycosyltransferase mutant according to claim 1.

3. A primer pair for amplifying the gene according to claim 2.

4. A recombinant vector comprising the gene according to claim 2 and expressing the glycosyltransferase mutant according to claim 1.

5. A recombinant cell comprising the recombinant vector according to claim 4 and expressing the glycosyltransferase mutant according to claim 1.

6. Use of the glycosyltransferase mutant according to claim 1 or the recombinant glycosyltransferase mutant produced by the recombinant cell according to claim 5 in catalyzing the biosynthesis of Reb D from Reb A.

7. A method for catalyzing the biosynthesis of Reb D from Reb A, characterized in that: The method comprises taking Reb A as a substrate, taking UDPG as a glucose donor, and utilizing the glycosyltransferase mutant according to claim 1 or the recombinant glycosyltransferase mutant produced by the recombinant cell according to claim 5 to carry out an enzymatic reaction to biosynthesize Reb D.

8. The method according to claim 7, characterized in that: The temperature of the enzymatic reaction is 40° C. and the time is 12 h.

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