A udp-glucosyltransferase mutant and a method for preparing rebaudioside d thereof

Abstract

This invention proposes a UDP-glucosyltransferase mutant and a method for preparing rebaudioside D. Site-directed mutagenesis of UDP-glucosyltransferase is performed, and the mutant plasmid is transferred to *E. coli* to obtain recombinant engineered bacteria. The recombinant engineered bacteria express the protein through fermentation to obtain a crude enzyme solution of the UDP-glucosyltransferase mutant. ADP-dependent enzyme activity and temperature-dependent enzyme activity of the UDP-glucosyltransferase mutant were measured. The mutant ADP-dependent enzyme activity was significantly increased, and it exhibited thermostability. Rebaudioside D was prepared using rebaudioside A, sucrose, ADP, the UDP-glucosyltransferase mutant, and AtSUS sucrose synthase hypercatalytic synthase.

Description

Technical Field

[0001] This invention relates to the field of steviol glycoside technology, and in particular to a UDP-glucosyltransferase mutant and a method for preparing rebaudioside D. Background Technology

[0002] High-sugar diets have led to a surge in metabolic diseases, such as obesity, diabetes, hypertension, and cardiovascular diseases, posing a serious threat to global public health and causing a rapid decline in individual quality of life. Therefore, the development of next-generation low-sugar or zero-calorie sweeteners has become urgent. Plant-derived steviol glycosides, due to their low calorie content, are considered a next-generation sweetener to replace high-calorie sugars. Rebaudioside D is a natural steviol glycoside compound with high sweetness and low calories, and is widely used in the food and beverage industry. With increasing consumer preference for low-sugar, low-calorie products, rebaudioside D has become one of the fastest-growing natural sweeteners in terms of global usage.

[0003] Glucosyltransferases are enzymes that transfer glucose residues during enzymatic reactions. Their mechanism of action involves catalyzing the transfer of glucose residues from a glycosyl donor to a glycosyl acceptor molecule, thereby regulating the activity of the acceptor molecule. UDP-glucosyltransferase (UGT) is a type of glucosyltransferase that uses UDP-glucose as a glycosyl donor and is present in almost all organisms.

[0004] UDP-glucose is short for uridine diphosphate glucose, also known as UDP-glucose or UDPG. It is a vitamin composed of uridine diphosphate and glucose, and can be regarded as "active glucose". It is widely distributed in the cells of plants, animals and microorganisms. It serves as a glucose donor in the synthesis of sucrose, starch, glycogen and other oligosaccharides and polysaccharides, and is the most common type of glycosyl donor.

[0005] Technical issues

[0006] With the widespread application of the natural sweetener steviol glycosides and the increasing development of biocatalysis technology, UDP-glucosyltransferases are increasingly used in the biocatalytic preparation of steviol glycosides. There are many types of UDP-glucosyltransferases. Currently, most of the enzymes used in the enzymatic preparation of steviol glycosides are wild-type enzymes derived from plant cells. UDP-glucosyltransferases can catalyze the conversion of rebaudioside A, sucrose, and UDP to rebaudioside D. However, in production, there are drawbacks such as the high price of UDP, low efficiency in catalyzing the conversion of rebaudioside A, sucrose, and ADP to rebaudioside D by wild-type UDP-glucosyltransferases, and low enzyme activity temperature. Therefore, it is necessary to modify wild-type UDP-glucosyltransferases to obtain modified enzymes with higher enzyme activity, ADP dependence, and adaptability to higher enzyme activity temperatures, in order to better serve large-scale industrial production.

[0007] Technical solutions

[0008] To address the aforementioned technical problems, this invention provides a UDP-glucosyltransferase mutant with higher enzyme activity, stronger ADP dependence, and a higher adaptation temperature for enzyme activity. This invention provides a UDP-glucosyltransferase mutant and a method for preparing rebaudidine D. The UDP-glucosyltransferase mutant catalyzes the synthesis of rebaudidine D from rebaudidine A, sucrose, and ADP.

[0009] As a preferred technical solution, the UDP-glucosyltransferase mutant is a mutation of the parental amino acid sequence number as shown in SEQ ID NO: 2, which is C31M / R310A / Q342W / F354G or Q36W / E299K / R310A / F354G or K290E / Q342W / F354G / L367I.

[0010] A method for preparing rebaudioside D from a UDP-glucosyltransferase mutant, using rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant as substrates, to catalyze a reaction to generate rebaudioside D.

[0011] As a preferred technical solution, rebaudin A, sucrose, ADP, AtSUS sucrose synthase and UDP-glucosyltransferase mutant are used as substrates, with a pH of 6.0-7.5, a temperature of 35℃-70℃, and a reaction time of 15min-1.5h to catalyze the reaction to generate rebaudin D.

[0012] As a preferred technical solution, rebaudine A, sucrose, ADP, AtSUS sucrose synthase and UDP-glucosyltransferase mutant are used as substrates, the pH is 7.0, the temperature is 60℃-70℃, the reaction time is 1.5h, and the catalytic reaction generates rebaudine D.

[0013] As a preferred technical solution, the nucleotide sequence of the UDP-glucosyltransferase mutant is shown in SEQ ID NO.3.

[0014] A method for preparing rebaudioside D using a UDP-glucosyltransferase mutant involves using rebaudioside A, sucrose, ADP, AtSUS sucrose synthase, and the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant as substrates to catalyze a reaction to generate rebaudioside D.

[0015] As a preferred technical solution, rebaudin A, sucrose, ADP, AtSUS sucrose synthase and UDP-glucosyltransferase mutant C31M / R310A / Q342W / F354G are used as substrates, the pH is 6.0-7.5, the temperature is 35℃-70℃, and the reaction time is 15min-1.5h to catalyze the reaction to generate rebaudin D.

[0016] As a preferred technical solution, rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant are used as substrates, the pH is 7.0, the temperature is 60℃, and the reaction time is 1.5h to catalyze the reaction to generate rebaudioside D.

[0017] A method for preparing rebaudioside D from a UDP-glucosyltransferase mutant is characterized by the use of 6.0 kg of rebaudioside A, 2.1 kg of sucrose, and 2.5 g of ADP, in a total volume of 30.0 L, along with 13.5 g of crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant and 8.1 g of AtSUS sucrose synthase, with the pH adjusted to 7.0. The reaction is carried out at 60 °C, catalyzing the formation of rebaudioside D.

[0018] Beneficial effects

[0019] 1. This invention provides a UDP-glucosyltransferase mutant, which can efficiently catalyze the conversion of rebaudioside A, sucrose and ADP into rebaudioside D.

[0020] 2. In this invention, the wild-type UDP-glucosyltransferase is mutated to C31M / R310A / Q342W / F354G or Q36W / E299K / R310A / F354G or K290E / Q342W / F354G / L367I.

[0021] 3. The UDP-glucosyltransferase mutant provided by this invention has high enzyme activity, with a substrate conversion rate of up to 92.5%.

[0022] 4. The UDP-glucosyltransferase mutant provided by this invention has high temperature adaptability and high conversion rate at 60℃-70℃.

[0023] 5. This invention provides 6.0 kg of rebaudioside A, 2.1 kg of sucrose, and 2.5 g of ADP, with a total volume of 30.0 L, along with 13.5 g of crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant and 8.1 g of AtSUS sucrose synthase, with the pH adjusted to 7.0. The reaction is carried out at 60 °C, catalyzing the production of rebaudioside D. After 2 hours of reaction, 90.1% conversion is achieved.

[0024] The best embodiment of the present invention

[0025] To facilitate understanding of the present invention, it will be described in detail below with reference to specific embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0026] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice of this invention, preferred methods and materials are now described.

[0027] TB medium: yeast extract 23.6 g / L, tryptone 11.8 g / L, dipotassium hydrogen phosphate 9.4 g / L, potassium dihydrogen phosphate 2.2 g / L, glycerol 4.0 mL / L.

[0028] LB medium: peptone 10.0 g / L, sodium chloride 10.0 g / L, yeast extract 5.0 g / L.

[0029] PCR reaction system: 5 μL of 10x high-fidelity buffer containing MgSO4, 1 μL of 10 mM dNTP mixture, 1 μL of upstream primer solution, 1 μL of downstream primer solution, 1 μL of template plasmid solution, 1.2 μL of high-fidelity DNA polymerase (2.5 U / μL), and 9.8 μL of ddH2O.

[0030] PCR setup program: initial denaturation 95℃ for 3 min, denaturation 95℃ for 30 s, annealing 60℃ for 1 min, extension 68℃ for 12 min, 18 cycles, final extension 68℃ for 10 min, storage at 12℃.

[0031] Example 1: UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant

[0032] 1. Preparation of plasminogenic granules

[0033] Based on the codon optimization of the amino acid sequence of UDP-glucosyltransferase from rice (Oryza sativa Japonica Group) (GenBank accession number: XP_015629141.1), the required gene fragment was synthesized and ligated into the pET28a vector with Nde I and Xho I restriction sites at both ends, respectively, to obtain the UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid. The obtained parental plasmid was then transformed into E. coli DH5α, and colonies were selected after Kana screening and sequencing. The nucleotide sequence of the cloned UDP-glucosyltransferase parent is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0034] 2. Site-directed mutagenesis of UDP-glucosyltransferase

[0035] (1) The wild-type UDP-glucosyltransferase (amino acid sequence as shown in SEQ ID NO.1) was mutated using the whole plasmid site-directed mutagenesis PCR method. The first round of site-directed mutagenesis PCR was performed using the UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid as a template and C31M_F / C31M_R primer pair. The primers were designed as follows:

[0036] C31M_F: '-CGGCCATCTGCTGCCGATGCTGGATCTGGCTCAG-3'

[0037] C31M_R: '-CTGAGCCAGATCCAGCATCGGCAGCAGATGGCCG-3'

[0038] After PCR, 1 μL of Dpn I enzyme was added and thoroughly mixed. The mixture was then incubated at 37°C for 1.0 h to digest the template plasmid. Subsequently, 5 μL of PCR reaction solution was added to DH5α competent cells, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42°C water bath for 60 s, immediately returned to ice for 3 min, and then cultured in a clean bench with 500 μL of antibiotic-free LB medium (37°C, 220 rpm, 60 min). The culture was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining 100 μL of cells was resuspended by pipetting. 100 μL of the culture was transferred to LB solid medium containing Kana resistance in a clean bench and gently spread with a sterile spreader. After absorption, the plates were inverted and incubated overnight at 37°C. A single colony from each plate was inoculated into 5.0 mL of medium containing 50 μg / L Kana. After culturing in LB medium (37℃, 220rpm, 6.0h), the sample was sent for sequencing. After sequencing, the service company returned the correct mutant plasmid pET-28a(+)-OsUGT(C31M).

[0039] (2) Using pET-28a(+)-OsUGT(C31M) as the template plasmid, and R310A_F / R310A_R as the primer pair, a second round of site-directed mutagenesis was performed. The primers were designed as follows:

[0040] R310A_F: '-GGCACCCGTTTCCTGTGGGCCCTGGCAAAACCGACCGGC GTGTCTGAC-3'

[0041] R310A_R: '-GTCAGACACGCCGGTCGGTTTTGCCAGGGCCCACAGGAA ACGGGTGCC-3'

[0042] The remaining steps are as follows (1), and the mutant plasmid pET-28a(+)-OsUGT(C31M / R310A) is obtained.

[0043] (3) Using pET-28a(+)-OsUGT(C31M / R310A) as the template plasmid, and Q342W_F / Q342W_R as the primer pair, a third round of site-directed mutagenesis was performed. The primers were designed as follows:

[0044] Q342W_F: '-GCAACCCGCTGGGTACCGTGGATGAGCATCCTGGCGCA TGCTGCTG-3'

[0045] Q342W_R: '-CAGCAGCATGCGCCAGGATGCTCATCCACGGTACCCAG CGGGTTGC-3'

[0046] The remaining steps are as follows (1), and the mutant plasmid pET-28a(+)-OsUGT(C31M / R310A / Q342W) is obtained.

[0047] (4) Using pET-28a(+)-OsUGT(C31M / R310A / Q342W) as the template plasmid, and F354G_F / F354G_R as the primer pair, a fourth round of site-directed mutagenesis was performed. The primers were designed as follows:

[0048] F354G_F: '-CGCATGCTGCTGTGGGCGCAGGTCTGACCCACTGTGGTT GGAAC-3'

[0049] F354G_R: '-GTTCCAACCACAGTGGGTCAGACCTGCGCCCACAGCAGC ATGCG-3'

[0050] The remaining steps are as follows (1), to obtain the mutant plasmid pET-28a(+)-OsUGT(C31M / R310A / Q342W / F354G), the nucleotide sequence of the mutant glucose dehydrogenase (C31M / R310A / Q342W / F354G) is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0051] 3. Preparation of UDP-glucosyltransferase enzyme solution

[0052] The mutant plasmid pET-28a(+)-OsUGT(C31M / R310A / Q342W / F354G) was transformed into E. coli BL21(DE3) to obtain the recombinant engineered strain E. coli BL21(DE3)-OsUGT(C31M / R310A / Q342W / F354G). Recombinant engineered E. coli BL21(DE3)-OsUGT (C31M / R310A / Q342W / F354G) was inoculated at a 1% ratio into 4.0 mL of liquid LB medium and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculation rate to a large volume of liquid LB medium and cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM–1 mM and cultured at 20–37°C with shaking for 12.0–16.0 h. After induction, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.2). The cells were then sonicated in an ice bath to disrupt their structure. The disrupted cells were centrifuged, and the supernatant was collected to obtain the crude enzyme solution containing the UDP-glucosyltransferase OsUGT (C31M / R310A / Q342W / F354G) mutant.

[0053] 4. ADP-dependent enzyme activity assay

[0054] Using 2.0 g of rebaudioside A as a substrate, 0.7 g of sucrose, 1.0 mg of ADP, and 3.0 mg of AtSUS sucrose synthase, 5.0 mg of crude enzyme solution of the UDP-glucosyltransferase OsUGT (C31M / R310A / Q342W / F354G) mutant prepared in Part 3 was added, bringing the total volume to 10.0 ml. The pH was adjusted to 7.2, and the reaction was carried out at 37℃ and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity. The enzyme activity was 670.6 ± 2.6 U / mg.

[0055] 5. Reaction temperature measurement

[0056] Eight portions of rebaudioside A (2.0 g), sucrose (0.7 g), ADP (1.0 mg), and AtSUS sucrose synthase (3.0 mg) were prepared. Then, 5.0 mg of crude enzyme solution of the UDP-glucosyltransferase OsUGT (C31M / R310A / Q342W / F354G) mutant prepared in Part 3 was added, with a total volume of 10.0 ml for each portion. The pH was adjusted to 7.2 with 10M sodium hydroxide. Temperatures were set at 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃. 50 μL of the reaction solution was collected at 5 min, 15 min, 30 min, and 1.0 h for HPLC analysis. The conversion rates of rebaudioside D at these temperatures are shown in the table below.

[0057] temperature 5min 15min 30min 1.0h 35℃ 5.6％ 21.9％ 44.3％ 66.6％ 40℃ 8.3％ 30.1％ 54.2％ 71.3％ 45℃ 15.1％ 32.9％ 63.1％ 79.4％ 50℃ 20.7％ 45.8％ 65.9％ 85.0％ 55℃ 32.1％ 54.2％ 74.6％ 88.2％ 60℃ 34.3％ 56.5％ 76.3％ 92.5％ 65℃ 25.9％ 47.4％ 72.6％ 88.5％ 70℃ 20.9％ 43.4％ 73.4％ 87.6％

[0058] Embodiments of the present invention

[0059] Example 2: UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant

[0060] 1. Preparation of plasminogenic granules

[0061] Based on the codon optimization of the amino acid sequence of UDP-glucosyltransferase from rice (Oryza sativa Japonica Group) (GenBank accession number: XP_015629141.1), the required gene fragment was synthesized and ligated into the pET28a vector with Nde I and Xho I restriction sites at both ends, respectively, to obtain the UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid. The obtained parental plasmid was then transformed into E. coli DH5α, and colonies were selected after Kana screening and sequencing. The nucleotide sequence of the cloned UDP-glucosyltransferase parent is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0062] 2. Site-directed mutagenesis of UDP-glucosyltransferase

[0063] (1) The wild-type UDP-glucosyltransferase (amino acid sequence as shown in SEQ ID NO.1) was mutated using the whole plasmid site-directed mutagenesis PCR method. The UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid was used as a template, and the first round of site-directed mutagenesis PCR was performed using Q36W_F / Q36W_R primer pair. The primers were designed as follows:

[0064] Q36W_F: '-TGCCGTGCCTGGATCTGGCTTGGCGTCTGGCTTCTCGCGG TCA-3'

[0065] Q36W_R: '-TGACCGCGAGAAGCCAGACGCCAAGCCAGATCCAGGCA CGGCA-3'

[0066] After PCR, 1 μL of Dpn I enzyme was added and thoroughly mixed. The mixture was then incubated at 37°C for 1.0 h to digest the template plasmid. Subsequently, 5 μL of PCR reaction solution was added to DH5α competent cells, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42°C water bath for 60 s, immediately returned to ice for 3 min, and then cultured in a clean bench with 500 μL of antibiotic-free LB medium (37°C, 220 rpm, 60 min). The culture was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining 100 μL of cells was resuspended by pipetting. 100 μL of the culture was transferred to LB solid medium containing Kana resistance in a clean bench and gently spread with a sterile spreader. After absorption, the plates were inverted and incubated overnight at 37°C. A single colony from each plate was inoculated into 5.0 mL of medium containing 50 μg / L Kana. After culturing in LB medium (37℃, 220rpm, 6.0h), the sample was sent for sequencing. After sequencing, the service company returned the correct mutant plasmid pET-28a(+)-OsUGT(Q36W).

[0067] (2) Using pET-28a(+)-OsUGT(Q36W) as the template plasmid, and E299K_F / E299K_R as the primer pair, a second round of site-directed mutagenesis was performed. The primers were designed as follows:

[0068] E299K_F: '-GGCACCCGTTTCCTGTGGGCCCTGGCAAAACCGACCGGC GTGTCTGAC-3'

[0069] E299K_R: '-GTCAGACACGCCGGTCGGTTTTGCCAGGGCCCACAGGAA ACGGGTGCC-3'

[0070] The remaining steps are as follows (1), and the mutant plasmid pET-28a(+)-OsUGT(Q36W / E299K) is obtained.

[0071] (3) Using pET-28a(+)-OsUGT(Q36W / E299K) as the template plasmid, and R310A_F / R310A_R as the primer pair, a third round of site-directed mutagenesis was performed. The primers were designed as follows:

[0072] R310A_F: '-GGCACCCGTTTCCTGTGGGCCCTGGCAAAACCGACCGGC GTGTCTGAC-3'

[0073] R310A_R: '-GTCAGACACGCCGGTCGGTTTTGCCAGGGCCCACAGGAA ACGGGTGCC-3'

[0074] The remaining steps are as follows (1), and the mutant plasmid pET-28a(+)-OsUGT(Q36W / E299K / R310A) is obtained.

[0075] (4) Using pET-28a(+)-OsUGT(Q36W / E299K / R310A) as the template plasmid, and F354G_F / F354G_R as the primer pair, a fourth round of site-directed mutagenesis was performed. The primers were designed as follows:

[0076] F354G_F: '-CGCATGCTGCTGTGGGCGCAGGTCTGACCCACTGTGGTT GGAAC-3'

[0077] F354G_R: '-GTTCCAACCACAGTGGGTCAGACCTGCGCCCACAGCAGC ATGCG-3'

[0078] The remaining steps are as follows (1), to obtain the mutant plasmid pET-28a(+)-OsUGT(Q36W / E299K / R310A / F354G), the nucleotide sequence of the mutated glucose dehydrogenase mutant is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0079] 3. Preparation of UDP-glucosyltransferase enzyme solution

[0080] The mutant plasmid pET-28a(+)-OsUGT(Q36W / E299K / R310A / F354G) was transformed into E. coli BL21(DE3) to obtain the recombinant engineered strain E. coli BL21(DE3)-OsUGT(Q36W / E299K / R310A / F354G). Recombinant engineered E. coli BL21(DE3)-OsUGT (Q36W / E299K / R310A / F354G) was inoculated at a 1% ratio into 4.0 mL of liquid LB medium and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculation rate to a large volume of liquid LB medium and cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM–1 mM and cultured at 20–37°C with shaking for 12.0–16.0 h. After induction, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.2). The cells were then sonicated in an ice bath, and the lysate was centrifuged. The supernatant was collected to obtain the crude enzyme solution containing UDP-glucosyltransferase OsUGT (Q36W / E299K / R310A / F354G).

[0081] 4. ADP-dependent enzyme activity assay

[0082] Using 2.0 g of rebaudioside A as a substrate, 0.7 g of sucrose, 1.0 mg of ADP, and 3.0 mg of AtSUS sucrose synthase, along with 5.0 mg of crude enzyme solution of the UDP-glucosyltransferase OsUGT (Q36W / E299K / R310A / F354G) mutant prepared in Part 3, the total volume was 10.0 ml. The pH was adjusted to 7.2, and the reaction was carried out at 37℃ and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity. The enzyme activity was 378.6 ± 1.9 U / mg.

[0083] 5. Reaction temperature measurement

[0084] Eight portions of rebaudioside A (2.0 g), sucrose (0.7 g), ADP (1.0 mg), and AtSUS sucrose synthase (3.0 mg) were prepared. Then, 5.0 mg of crude enzyme solution of the UDP-glucosyltransferase OsUGT (Q36W / E299K / R310A / F354G) mutant prepared in Part 3 was added to each portion, with a total volume of 10.0 ml. The pH was adjusted to 7.2 with 10M sodium hydroxide. Temperatures were set at 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃. 50 μL of the reaction solution was collected at 5 min, 15 min, 30 min, and 1.0 h for HPLC analysis. The conversion rates of rebaudioside D at these temperatures are shown in the table below.

[0085] temperature 5min 15min 30min 1.0h 35℃ 5.0％ 16.4％ 31.3％ 54.1％ 40℃ 6.7％ 20.9％ 33.1％ 55.2％ 45℃ 8.1％ 23.7％ 35.9％ 57.2％ 50℃ 8.7％ 23.2％ 38.4％ 60.7％ 55℃ 12.3％ 25.2％ 45.8％ 65.3％ 60℃ 16.8％ 28.3％ 48.1％ 69.3％ 65℃ 17.2％ 28.6％ 50.3％ 73.6％ 70℃ 18.1％ 30.2％ 52.7％ 74.2％

[0086] Example 3: UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant

[0087] 1. Preparation of plasminogenic granules

[0088] Based on the codon optimization of the amino acid sequence of UDP-glucosyltransferase from rice (Oryza sativa Japonica Group) (GenBank accession number: XP_015629141.1), the required gene fragment was synthesized and ligated into the pET28a vector with Nde I and Xho I restriction sites at both ends, respectively, to obtain the UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid. The obtained parental plasmid was then transformed into E. coli DH5α, and colonies were selected after Kana screening and sequencing. The nucleotide sequence of the cloned UDP-glucosyltransferase parent is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0089] 2. Site-directed mutagenesis of UDP-glucosyltransferase

[0090] (1) The wild-type UDP-glucosyltransferase (amino acid sequence as shown in SEQ ID NO.1) was mutated using the whole plasmid site-directed mutagenesis PCR method. The UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid was used as a template, and the first round of site-directed mutagenesis PCR was performed using K290E__F / K290E__R primer pair. The primers were designed as follows:

[0091] K290E_F: '-AGGTGCCGCTGGGTGTTGAAGAAGTACACGAGCTGGCGC TGGGTC-3'

[0092] K290E_R: '-GACCCAGCGCCAGCTCGTGTACTTCTTCACACCCAGCG GCACCT-3'

[0093] After PCR, 1 μL of Dpn I enzyme was added and thoroughly mixed. The mixture was then incubated at 37°C for 1.0 h to digest the template plasmid. Subsequently, 5 μL of PCR reaction solution was added to DH5α competent cells, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42°C water bath for 60 s, immediately returned to ice for 3 min, and then cultured in a clean bench with 500 μL of antibiotic-free LB medium (37°C, 220 rpm, 60 min). The culture was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining 100 μL of cells was resuspended by pipetting. 100 μL of the culture was transferred to LB solid medium containing Kana resistance in a clean bench and gently spread with a sterile spreader. After absorption, the plates were inverted and incubated overnight at 37°C. A single colony from each plate was inoculated into 5.0 mL of medium containing 50 μg / L Kana. After culturing in LB medium (37℃, 220rpm, 6.0h), the sample was sent for sequencing. After sequencing, the service company returned the correct mutant plasmid pET-28a(+)-OsUGT(K290E).

[0094] (2) Using pET-28a(+)-OsUGT(K290E) as the template plasmid, and Q342W_F / Q342W_R as the primer pair, a second round of site-directed mutagenesis was performed. The primers were designed as follows:

[0095] Q342W_F: '-GCAACCCGCTGGGTACCGTGGATGAGCATCCTGGCGCA TGCTGCTG-3'

[0096] Q342W_R: '-CAGCAGCATGCGCCAGGATGCTCATCCACGGTACCCAG CGGGTTGC-3'

[0097] The remaining steps are as follows (1), and the mutant plasmid pET-28a(+)-OsUGT(K290E / Q342W) is obtained.

[0098] (3) Using pET-28a(+)-OsUGT(K290E / Q342W) as the template plasmid, and F354G_F / F354G_R as the primer pair, a third round of site-directed mutagenesis was performed. The primers were designed as follows:

[0099] F354G_F: '-CGCATGCTGCTGTGGGCGCAGGTCTGACCCACTGTGGTT GGAAC-3'

[0100] F354GR: '-GTTCCAACCACAGTGGGTCAGACCTGCGCCCACAGCAGC ATGCG-3'

[0101] The remaining steps are as follows (1), and the mutant plasmid pET-28a(+)-OsUGT(K290E / Q342W / F354G) is obtained.

[0102] (4) Using pET-28a(+)-OsUGT(K290E / Q342W / F354G) as the template plasmid, and L367I_F / L367I_R as the primer pair, a fourth round of site-directed mutagenesis was performed. The primers were designed as follows:

[0103] L367I_F: '-GGAACTCCACTATCGAAGGCATCATGTTCGGTCACCCTCT GATTATGCTGCCG-3'

[0104] L367I_R: '-CGGCAGCATAATCAGAGGGTGACCGAACATGATGCCTTC GATAGTGGAGTTCC-3'

[0105] The remaining steps are as follows (1), and the mutant plasmid pET-28a(+)-OsUGT(K290E / Q342W / F354G / L367I) is obtained. The nucleotide sequence of the mutated glucose dehydrogenase mutant is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8.

[0106] 3. Preparation of UDP-glucosyltransferase enzyme solution

[0107] The mutant plasmid pET-28a(+)-OsUGT(K290E / Q342W / F354G / L367I) was transformed into E. coli BL21(DE3) to obtain the recombinant engineered strain E. coli BL21(DE3)-OsUGT(K290E / Q342W / F354G / L367I). Recombinant engineered E. coli BL21(DE3)-OsUGT(K290E / Q342W / F354G / L367I) was inoculated at a 1% ratio into 4.0 mL of liquid LB medium and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculation rate to a large volume of liquid LB medium and cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM–1 mM and cultured at 20–37°C with shaking for 12.0–16.0 h. After induction, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.2). The cells were then sonicated in an ice bath, and the lysate was centrifuged. The supernatant was collected to obtain the crude enzyme solution containing UDP-glucosyltransferase OsUGT(K290E / Q342W / F354G / L367I).

[0108] 4. ADP-dependent enzyme activity assay

[0109] Using 2.0 g of rebaudioside A as a substrate, 0.7 g of sucrose, 1 mg of ADP, and 3.0 mg of AtSUS sucrose synthase, 5.0 mg of crude enzyme solution of the UDP-glucosyltransferase OsUGT (K290E / Q342W / F354G / L367I) mutant prepared in Part 3 was added to each sample, with a total volume of 10.0 ml. The pH was adjusted to 7.2, and the reaction was carried out at 37℃ and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity. The enzyme activity was 523.6 ± 3.2 U / mg.

[0110] 5. Reaction temperature measurement

[0111] Eight portions of rebaudioside A (2.0 g), sucrose (0.7 g), ADP (1.0 mg), and AtSUS sucrose synthase (3.0 mg) were prepared. Then, 5.0 mg of crude enzyme solution of the UDP-glucosyltransferase OsUGT (K290E / Q342W / F354G / L367I) mutant prepared in Part 3 was added to each portion, with a total volume of 10.0 ml. The pH was adjusted to 7.2 with 10M sodium hydroxide. The temperatures were 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃. 50 μL of the reaction solution was collected at 5 min, 15 min, 30 min, and 1.0 h for HPLC analysis. The conversion rates of rebaudioside D at these temperatures are shown in the table below.

[0112] temperature 5min 15min 30min 1.0h 35℃ 5.9％ 19.7％ 40.7％ 63.3％ 40℃ 6.2％ 20.8％ 42.1％ 64.5％ 45℃ 6.2％ 20.1％ 42.3％ 64.2％ 50℃ 6.9％ 23.4％ 42.9％ 65.2％ 55℃ 7.1％ 24.3％ 42.7％ 67.1％ 60℃ 7.2％ 25.1％ 43.4％ 67.4％ 65℃ 8.8％ 25.3％ 43.9％ 68.7％ 70℃ 8.6％ 25.2％ 45.3％ 68.4％

[0113] Comparative Example 1: ADP-dependent enzyme activity assay of the parent UDP-glucosyltransferase

[0114] 1. Preparation of plasminogenic granules

[0115] The required gene fragment was synthesized based on the amino acid sequence of UDP-glucosyltransferase from rice (Oryza sativa Japonica Group) (GenBank accession number: XP_015629141.1), ligated into the pET28a vector, with Nde I and Xho I restriction sites at both ends, to obtain the UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid. The obtained parental plasmid was then transformed into E. coli DH5α, and colonies were selected after Kana screening and sequencing. The nucleotide sequence of the cloned UDP-glucosyltransferase parent is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0116] 2. Preparation of UDP-glucosyltransferase enzyme solution

[0117] The parental plasmid pET-28a(+)-OsUGT of UDP-glucosyltransferase was transformed into E. coli BL21(DE3) to obtain recombinant engineered E. coli BL21(DE3)-OsUGT. The recombinant engineered E. coli BL21(DE3)-OsUGT was inoculated at a ratio of 1% into 4.0 mL of liquid LB medium and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculation rate to a large volume of liquid LB medium and cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM–1.0 mM and cultured at 20–37°C with shaking for 12.0–16.0 h. After induction, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.2). The cells were then sonicated in an ice bath to disrupt them. The disrupted solution was centrifuged, and the supernatant was collected to obtain the crude enzyme solution containing the UDP-glucosyltransferase parent.

[0118] 3. ADP-dependent enzyme activity assay

[0119] Using 2.0 g of rebaudioside A as a substrate, 0.7 g of sucrose, 1.0 mg of ADP, and 3.0 mg of AtSUS sucrose synthase, along with 5.0 mg of crude enzyme solution of the UDP-glucosyltransferase parent prepared in Part 2, the total volume was 10.0 ml. The pH was adjusted to 7.2, and the reaction was carried out at 37 °C and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity, which was 247.67 ± 2.3 U / mg.

[0120] 4. Reaction temperature measurement

[0121] Eight portions of 2.0 g rebaudioside A, 0.7 mg sucrose, 1.0 mg ADP, and 3.0 mg AtSUS sucrose synthase were prepared, along with 5.0 mg of crude enzyme solution of the UDP-glucosyltransferase parent prepared in Part 2. Each portion had a total volume of 10.0 ml. The pH was adjusted to 7.2 using 10 M sodium hydroxide. Temperatures were set at 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃. 50 μL of the reaction solution was collected at 5 min, 15 min, 30 min, and 1.0 h for HPLC analysis. The conversion rates of rebaudioside D at these temperatures are shown in the table below.

[0122] temperature 5min 15min 30min 1.0h 35℃ 5.4％ 8.9％ 20.6％ 27.8％ 40℃ 7.3％ 10.3％ 23.7％ 40.6％ 45℃ 9.2％ 12.1％ 25.3％ 42.8％ 50℃ 9.0％ 11.5％ 24.0％ 41.9％ 55℃ 7.9％ 11.7％ 23.5％ 40.1％ 60℃ 6.8％ 10.9％ 21.7％ 38.7％ 65℃ 5.6％ 9.4％ 20.4％ 35.6％ 70℃ 4.0％ 8.9％ 18.7％ 33.7％

[0123] Example 4: Preparation of Rebaudioside D from UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant

[0124] 1. Optimization of reaction pH

[0125] Rebaudioside A 2.0g, sucrose 0.7g, and ADP 1.0mg were dissolved completely by stirring. Then, 5.0mg of crude enzyme solution of UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10.0ml for each sample. The pH was adjusted to 6.0, 6.5, 7.0, and 7.5. The reaction was carried out at 60℃ and 200rpm for 1.0h. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. The conversion rate of rebaudioside D was 83.2% at pH 6.0, 86.3% at pH 6.5, 91.8% at pH 7.0, and 85.7% at pH 7.5. The optimal pH for the reaction is 7.0.

[0126] 2. Optimization of reaction time

[0127] Rebaudioside A 2.0g, sucrose 0.7g, and ADP 1.0mg were dissolved completely by stirring. Then, 5.0mg of crude enzyme solution of UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10.0ml for each sample. The pH was adjusted to 7.2. The reaction was carried out at 60℃ and 200rpm for 15min, 30min, 1.0h, and 1.5h, respectively. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. The conversion rate of rebaudioside D was 53.9% when the reaction time was 15 min; 73.1% when the reaction time was 30 min; 91.3% when the reaction time was 1.0 h; and 91.4% when the reaction time was 1.5 h.

[0128] 3. Optimization of substrate feeding amount

[0129] Rebaudioside A 1.6g, 2.0g, 2.4g, and 3.0g, sucrose 0.7g, and ADP 1.0mg were prepared separately and stirred until completely dissolved. Then, 5.0mg of crude enzyme solution of UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10.0ml for each sample. The pH was adjusted to 7.2. The reactions were carried out at 60℃ and 200rpm for 1.0h. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. When rebaudioside A was 1.6 g, the conversion rate of rebaudioside D was 86.3%; when rebaudioside A was 2.0 g, the conversion rate of rebaudioside D was 91.7%; when rebaudioside A was 2.4 g, the conversion rate of rebaudioside D was 91.8%; and when rebaudioside A was 3.0 g, the conversion rate of rebaudioside D was 91.8%.

[0130] 4. Optimization of UDP-glucosyltransferase dosage

[0131] Prepare a solution by mixing 2.0 g of rebaudioside A, 0.7 g of sucrose, and 1.0 mg of ADP, stirring until completely dissolved. Add crude enzyme solutions of UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutants at concentrations of 4.0 mg, 6.0 mg, 8.0 mg, 10.0 mg, and 3.0 mg of AtSUS sucrose synthase, with a total volume of 10.0 ml for each sample. Adjust the pH to 7.2. React at 60℃ and 200 rpm for 1.0 h. After the reaction, take 50 μL of the reaction solution, filter through a 0.22 μm microfiltration membrane, and use as a liquid phase sample for HPLC analysis. When the amount of crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant was 4.0 mg, the conversion rate of rebaudioside D was 88.4%; when the amount of crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant was 6.0 mg, the conversion rate of rebaudioside D was 90.1%; when the amount of crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant was 8.0 mg, the conversion rate of rebaudioside D was 91.3%; and when the amount of crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant was 10.0 mg, the conversion rate of rebaudioside D was 91.3%. The optimal amount of crude enzyme solution for the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant is 8.0 mg.

[0132] Example 5: Preparation of Rebaudioside D from UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant

[0133] 1. Optimization of reaction pH

[0134] Rebaudioside A 2.0g, sucrose 0.7g, and ADP 1.0mg were dissolved completely by stirring. Then, 5.0mg of crude enzyme solution of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10.0ml for each sample. The pH was adjusted to 6.0, 6.5, 7.0, and 7.5. The reaction was carried out at 70℃ and 200rpm for 1h. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. The conversion rate of rebaudioside D was 65.4% at pH 6.0, 69.3% at pH 6.5, 72.3% at pH 7.0, and 68.0% at pH 7.5. The optimal pH for the reaction is 7.0.

[0135] 2. Optimization of reaction time

[0136] Rebaudioside A 2.0g, sucrose 0.7g, and ADP 1.0mg were dissolved completely by stirring. Then, 5.0mg of crude enzyme solution of the UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10.0ml for each sample. The pH was adjusted to 7.2. The reaction was carried out at 70℃ and 200rpm for 15min, 30min, 1.0h, and 1.5h, respectively. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. The conversion rate of rebaudioside D was 28.6% when the reaction time was 15 min; 50.1% when the reaction time was 30 min; 68.3% when the reaction time was 1.0 h; and 68.5% when the reaction time was 1.5 h.

[0137] 3. Optimization of substrate feeding amount

[0138] Rebaudioside A 1.6g, 2.0g, 2.4g, and 3.0g, sucrose 0.7g, and ADP 1.0mg were prepared separately and stirred until completely dissolved. Then, 5.0mg of crude enzyme solution of the UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10.0ml for each sample. The pH was adjusted to 7.2. The reactions were carried out at 70℃ and 200rpm for 1.0h. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. When rebaudioside A was 1.6 g, the conversion rate of rebaudioside D was 68.5%; when rebaudioside A was 2.0 g, the conversion rate of rebaudioside D was 72.0%; when rebaudioside A was 2.4 g, the conversion rate of rebaudioside D was 72.2%; and when rebaudioside A was 3.0 g, the conversion rate of rebaudioside D was 72.3%.

[0139] 4. Optimization of UDP-glucosyltransferase dosage

[0140] Prepare a solution by mixing 2.0 g of rebaudioside A, 0.7 g of sucrose, and 1.0 mg of ADP, stirring until completely dissolved. Add crude enzyme solutions of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant at concentrations of 4.0 mg, 6.0 mg, 8.0 mg, 10.0 mg, and 3.0 mg of AtSUS sucrose synthase, with a total volume of 10.0 ml for each sample. Adjust the pH to 7.2. Incubate the reaction at 70 °C and 200 rpm for 1.0 h. After the reaction, take 50 μL of the reaction solution, filter it through a 0.22 μm microfiltration membrane, and use it as a liquid phase sample for HPLC analysis. The conversion rate of rebaudioside D was 70.9% when the amount of crude enzyme solution of the UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant was 6.0 mg; the conversion rate was 73.4% when the amount of crude enzyme solution of the UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant was 8.0 mg; and the conversion rate was 73.4% when the amount of crude enzyme solution of the UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant was 10.0 mg.

[0141] Example 6: Preparation of Rebaudioside D from UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant

[0142] 1. Optimization of reaction pH

[0143] Rebaudioside A 2.0g, sucrose 0.7g, and ADP 1.0mg were dissolved completely by stirring. Then, 5.0mg of crude enzyme solution of the UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10.0ml for each sample. The pH was adjusted to 6.0, 6.5, 7.0, and 7.5. The reaction was carried out at 65℃ and 200rpm for 1.0h. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. The conversion rate of rebaudioside D was 64.2% at pH 6.0, 66.3% at pH 6.5, 67.8% at pH 7.0, and 67.0% at pH 7.5. The optimal pH value for the reaction is 7.0.

[0144] 2. Optimization of reaction time

[0145] Rebaudioside A 2.0g, sucrose 0.7g, and ADP 1.0mg were dissolved completely by stirring. Then, 5.0mg of crude enzyme solution of the UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10ml per batch. The pH was adjusted to 7.2. The reaction was carried out at 65℃ and 200rpm for 15min, 30min, 1.0h, and 1.5h, respectively. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. The conversion rate of rebaudioside D was 22.9% when the reaction time was 15 min; 40.2% when the reaction time was 30 min; 67.3% when the reaction time was 1.0 h; and 68.5% when the reaction time was 1.5 h.

[0146] 3. Optimization of substrate feeding amount

[0147] Rebaudioside A 1.6g, 2.0g, 2.4g, and 3.0g, sucrose 0.7g, and ADP 1.0mg were prepared separately and stirred until completely dissolved. Then, 5.0mg of crude enzyme solution of the UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant and 3.0mg of AtSUS sucrose synthase were added, with a total volume of 10.0ml for each sample. The pH was adjusted to 7.2. The reactions were carried out at 65℃ and 200rpm for 1.0h. After the reaction, 50μL of the reaction solution was filtered through a 0.22μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. When 1.6 mg of rebaudioside A was used, the conversion rate of rebaudioside D was 65.7%; when 2.0 g of rebaudioside A was used, the conversion rate of rebaudioside D was 67.3%; when 2.4 g of rebaudioside A was used, the conversion rate of rebaudioside D was 67.4%; and when 3.0 g of rebaudioside A was used, the conversion rate of rebaudioside D was 67.4%.

[0148] 4. Optimization of UDP-glucosyltransferase dosage

[0149] Prepare a solution by mixing 2.0 g of rebaudioside A, 0.7 g of sucrose, and 1 mg of ADP, stirring until completely dissolved. Add crude enzyme solutions of UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant at concentrations of 4.0 mg, 6.0 mg, 8.0 mg, 10.0 mg, and 3.0 mg of AtSUS sucrose synthase, with a total volume of 10 ml for each sample. Adjust the pH to 7.2. React at 65℃ and 200 rpm for 1.0 h. After the reaction, take 50 μL of the reaction solution, filter through a 0.22 μm microfiltration membrane, and use as a liquid phase sample for HPLC analysis. The conversion rate of rebaudioside D was 66.4% when the amount of crude enzyme solution of the UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant was 4.0 mg; 68.5% when the amount of crude enzyme solution of the UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant was 6.0 mg; 68.8% when the amount of crude enzyme solution of the UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant was 8.0 mg; and 68.8% when the amount of crude enzyme solution of the UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant was 10.0 mg.

[0150] Industrial applicability

[0151] Rebaudioside A (6.0 kg), sucrose (2.1 g), and ADP (2.5 g) were mixed in a total volume of 30.0 L and stirred until completely dissolved. Then, 13.5 g of crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant and 8.1 g of AtSUS sucrose synthase were added, and the pH was adjusted to 7.0. The reaction was carried out at 60 °C. After the reaction, 50 μL of the reaction solution was filtered through a 0.22 μm microfiltration membrane and used as a liquid phase sample for HPLC analysis. After 2 hours of reaction, 90.1% conversion was achieved.

[0152] Sequence List Free Content

[0153] SEQ ID NO.1 nucleotide sequence of UDP-glucosyltransferase from rice (Oryza sativa Japonica Group).

[0154] SEQ ID NO.2 Rice (Oryza sativa Japonica Group) UDP-glucosyltransferase amino acid sequence.

[0155] SEQ ID NO.3 Nucleotide sequence of rice (Oryza sativa Japonica Group) UDP-glucosyltransferase C31M / R310A / Q342W / F354G after mutation.

[0156] SEQ ID NO.4 Amino acid sequence of rice (Oryza sativa Japonica Group) UDP-glucosyltransferase C31M / R310A / Q342W / F354G after mutation.

[0157] SEQ ID NO.5 Nucleotide sequence of rice (Oryza sativa Japonica Group) UDP-glucosyltransferase Q36W / E299K / R310A / F354G after mutation.

[0158] SEQ ID NO.6 Amino acid sequence of rice (Oryza sativa Japonica Group) UDP-glucosyltransferase Q36W / E299K / R310A / F354G after mutation.

[0159] SEQ ID NO.7 Nucleotide sequence of rice (Oryza sativa Japonica Group) UDP-glucosyltransferase K290E / Q342W / F354G / L367I after mutation.

[0160] SEQ ID NO.8 Amino acid sequence of rice (Oryza sativa Japonica Group) UDP-glucosyltransferase K290E / Q342W / F354G / L367I after mutation.

Claims

1. A UDP-glucosyltransferase mutant, characterized in that, The UDP-glucosyltransferase mutant catalyzes the formation of rebaudioside D from rebaudioside A, sucrose, and ADP; the UDP-glucosyltransferase mutant is a mutation of the parental amino acid sequence number as shown in SEQ ID NO: 2, to C31M / R310A / Q342W / F354G or Q36W / E299K / R310A / F354G or K290E / Q342W / F354G / L367I.

2. The UDP-glucosyltransferase mutant according to claim 1, characterized in that, The nucleotide sequence of the UDP-glucosyltransferase mutant is shown in SEQ ID NO.

3.

3. A method for preparing rebaudioside D from a UDP-glucosyltransferase mutant, characterized in that, Using rebaudine A, sucrose, ADP, AtSUS sucrose synthase, and the UDP-glucosyltransferase mutant as described in claim 1 as substrates, a catalytic reaction is carried out to generate rebaudine D.

4. The method for preparing rebaudioside D from a UDP-glucosyltransferase mutant according to claim 3, characterized in that, Using rebaudin A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant as described in claim 1 as substrates, the reaction was carried out at a pH of 6.0-7.5, a temperature of 35℃-70℃, and a reaction time of 15 min-1.5 h to catalyze the formation of rebaudin D.

5. The method for preparing rebaudioside D from a UDP-glucosyltransferase mutant according to claim 4, characterized in that, Using rebaudin A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant as described in claim 1 as substrates, the reaction was carried out at pH 7.0, temperature 60℃-70℃, and reaction time 1.5 h to catalyze the formation of rebaudin D.

6. A method for preparing rebaudioside D from a UDP-glucosyltransferase mutant, characterized in that, Using rebaudine A, sucrose, ADP, AtSUS sucrose synthase, and the UDP-glucosyltransferase mutant as described in claim 2 as substrates, a catalytic reaction is carried out to generate rebaudine D.

7. The method for preparing rebaudioside D from a UDP-glucosyltransferase mutant according to claim 6, characterized in that, Using rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant as described in claim 2 as substrates, the reaction was carried out at a pH of 6.0-7.5, a temperature of 35℃-70℃, and a reaction time of 15 min-1.5 h to catalyze the formation of rebaudioside D.

8. The method for preparing rebaudioside D from a UDP-glucosyltransferase mutant according to claim 7, characterized in that, Using rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant as described in claim 2 as substrates, the reaction was carried out at pH 7.0, temperature 60°C, and reaction time 1.5 h to catalyze the formation of rebaudioside D.

9. A method for preparing rebaudioside D from a UDP-glucosyltransferase mutant, characterized in that, Rebaudioside A 6.0 kg, sucrose 2.1 kg, ADP 2.5 g, total volume 30.0 L, crude enzyme solution of the UDP-glucosyltransferase mutant as described in claim 2 13.5 g, and AtSUS sucrose synthase 8.1 g, pH adjusted to 7.0, reaction carried out at 60 °C, catalyzing the reaction to produce rebaudioside D.