UDP-glucosyltransferase mutant and method for preparing rebaudioside D by using UDP-glucosyltransferase mutant

Abstract

The invention provides a UDP-glucosyltransferase mutant and a method for preparing rebaudioside D. The UDP-glucosyltransferase mutant is subjected to site-directed mutagenesis, mutant plasmid is transferred to escherichia coli to obtain recombinant engineering bacteria, the recombinant engineering bacteria are subjected to protein expression through fermentation, and UDP-glucosyltransferase mutant crude enzyme is obtained. The ADP-dependent enzyme activity and temperature of the UDP-glucosyltransferase mutant are measured, the ADP-dependent enzyme activity after mutation is remarkably improved, and the mutant has high temperature resistance. The rebaudioside D is prepared from rebaudioside A, sucrose, ADP (adenosine diphosphate), a UDP-glucosyltransferase mutant and AtSUS sucrose synthetase high-catalysis synthetase.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of steviol glycosides, in particular to a UDP-glucosyltransferase mutant and a method for preparing rebaudioside D by using the same. BACKGROUND

[0002] Due to high-sugar diet, more and more people suffer from different metabolic diseases such as obesity, diabetes, hypertension and cardiovascular diseases, which seriously threaten global public health and lead to rapid decline in the quality of life of individuals. Therefore, it is urgent to develop a new generation of low-sugar or zero-calorie sweeteners, and plant-derived steviol glycosides are considered to be the next generation of new sweeteners to replace high-calorie sugar because of their low calories. 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 the preference of consumers for low-sugar and low-calorie products, rebaudioside D has become one of the fastest growing natural sweeteners in the world.

[0003] Glucosyltransferase is an enzyme that transfers glucosyl in an enzyme reaction. The mechanism of action of the enzyme is to catalyze the transfer of glucose residues of a sugar donor to a sugar acceptor molecule, thereby regulating the activity of the acceptor molecule. UDP-glucosyltransferase (UGT) is a kind of glucosyltransferase, which uses UDP-glucose as a sugar donor and exists in almost all organisms.

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

[0005] TECHNICAL PROBLEM

[0006] Now, with the wide application of natural sweetener steviol glycoside and the increasing development of biocatalysis technology, UDP-glucosyltransferase is more and more applied in the field of biocatalytic preparation of steviol glycoside. There are many kinds of UDP-glucosyltransferase, and at present, the enzymes used in the field of biocatalytic preparation of steviol glycoside are mostly wild enzymes derived from plant cells. UDP-glucosyltransferase can catalyze rebaudioside A, sucrose and UDP to generate rebaudioside D. However, in production, the price of UDP is relatively high, and the wild enzyme of UDP-glucosyltransferase has low efficiency in catalyzing rebaudioside A, sucrose and ADP to generate rebaudioside D, low enzyme activity temperature and other shortcomings. Therefore, it is necessary to modify the wild enzyme of UDP-glucosyltransferase, so as to obtain modified enzyme with higher enzyme activity, ADP dependence and high enzyme activity temperature, so as to better serve industrialized mass production.

[0007] Technical solutions

[0008] In order to solve the above technical problems, a UDP-glucosyltransferase mutant with higher enzyme activity, stronger ADP dependence and higher enzyme activity temperature is provided. The present application provides a UDP-glucosyltransferase mutant and a method for preparing rebaudioside D. The UDP-glucosyltransferase mutant catalyzes rebaudioside A, sucrose and ADP to generate rebaudioside D.

[0009] As a preferred technical solution, the UDP-glucosyltransferase mutant is mutated from the parent amino acid sequence shown in SEQ ID NO: 2 to C31M / R310A / Q342W / F354G or Q36W / E299K / R310A / F354G or K290E / Q342W / F354G / L367I.

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

[0011] As a preferred technical solution, rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and UDP-glucosyltransferase mutant are used as substrates, the pH is 6.0-7.5, the temperature is 35℃-70℃, the reaction time is 15min-1.5h, and the catalytic reaction generates rebaudioside D.

[0012] As a preferred technical solution, rebaudioside 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 rebaudioside D.

[0013] As a preferred technical scheme, the nucleotide sequence of the UDP-glucose transferase mutant is shown as SEQ ID NO. 3.

[0014] A method for preparing rebaudioside D by a UDP-glucose transferase mutant, wherein rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucose transferase C31M / R310A / Q342W / F354G mutant are used as substrates to catalyze the reaction to generate rebaudioside D.

[0015] As a preferred technical scheme, rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucose transferase mutant C31M / R310A / Q342W / F354G are used as substrates, the pH is 6.0-7.5, the temperature is 35℃-70℃, the reaction time is 15min-1.5h, and the catalytic reaction generates rebaudioside D.

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

[0017] A method for preparing rebaudioside D by a UDP-glucose transferase mutant, wherein rebaudioside A 6.0kg, sucrose 2.1kg, ADP 2.5g, and a total volume of 30.0L are used, crude enzyme solution of the UDP-glucose transferase C31M / R310A / Q342W / F354G mutant 13.5g and AtSUS sucrose synthase 8.1g are used, the pH is adjusted to 7.0, and the catalytic reaction generates rebaudioside D under the condition of 60℃.

[0018] Beneficial effects

[0019] 1. The present application provides a UDP-glucose transferase mutant, and the mutant UDP-glucose transferase can catalyze rebaudioside A, sucrose and ADP to generate rebaudioside D with high efficiency.

[0020] 2. The wild-type UDP-glucose transferase is mutated to C31M / R310A / Q342W / F354G or Q36W / E299K / R310A / F354G or K290E / Q342W / F354G / L367I.

[0021] 3. The UDP-glucose transferase mutant provided by the present application has high enzyme activity, and the substrate conversion rate can reach 92.5% at most.

[0022] 4、The UDP-glucosyltransferase mutant provided by the application has higher temperature applicability, and has higher conversion rate at 60-70 DEG C.

[0023] 5、The application provides 6.0kg of rebaudioside A, 2.1kg of sucrose, 2.5g of ADP, a total volume of 30.0L, 13.5g of a crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant, and 8.1g of AtSUS sucrose synthase, and the pH is adjusted to 7.0. The reaction is carried out at 60 DEG C, and rebaudioside D is generated by catalysis. After 2h of reaction, 90.1% conversion is achieved.

[0024] Best mode of the application

[0025] In order to make the application easy to understand, specific examples will be combined below to describe the application in detail. However, before the detailed description of the application, it should be understood that the application is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for the purpose of describing the specific embodiments and are not 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 application belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice of the present application, the preferred methods and materials are now described.

[0027] TB medium: 23.6g / L of yeast powder, 11.8g / L of tryptone, 9.4g / L of potassium phosphate dibasic, 2.2g / L of potassium phosphate monobasic, and 4.0mL / L of glycerol.

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

[0029] PCR reaction system: 5ul of 10x high-fidelity buffer containing MgSO4, 1ul of 10mM dNTP mixture, 1ul of upstream primer solution, 1ul of downstream primer solution, 1ul of template plasmid solution, 1.2ul of high-fidelity DNA polymerase, 2.5U / ul, and 39.8ul of ddH2O.

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

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

[0032] 1. Preparation of parent plasmid

[0033] The desired gene fragment was synthesized according to the codon optimization of the amino acid sequence of UDP-glucosyltransferase derived from rice (Oryza sativa Japonica Group) (GenBank Accession No. XP_015629141.1), and was connected to the pET28a vector, with Nde I and Xho I as the enzyme cutting sites at both ends, to obtain the UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid. The obtained parent plasmid was then transformed into Escherichia coli DH5α, and after Kana screening, colonies were picked for sequencing to determine the nucleotide sequence of the cloned UDP-glucosyltransferase parent, which is shown as SEQ ID NO: 1, and the amino acid sequence thereof is shown as SEQ ID NO: 2.

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

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

[0036] C31M_F: '-CGGCCATCTGCTGCCGATGCTGGATCTGGCTCAG-3'

[0037] C31M_R: '-CTGAGCCAGATCCAGCATCGGCAGCAGATGGCCG-3'

[0038] After PCR, 1 μL Dpn I enzyme was added, mixed thoroughly, and then incubated at 37°C for 1.0 h to digest the template plasmid. Then 5 μL of the PCR reaction solution was added to DH5α competent cells, which were placed on ice for 30 min, heated at 42°C for 60 s in a water bath, immediately placed on ice for 3 min, and then added to 500 μL of LB medium without antibiotics in a clean bench and cultured (37°C, 220 rpm, 60 min). The bacterial solution was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining 100 μL of bacterial solution was resuspended by blowing and sucking with a syringe tip, and then 100 μL of the bacterial solution was removed with a pipette gun and added to LB solid medium containing Kana. The medium was evenly coated with a sterile coating rod, and after the bacterial solution was absorbed, the plate was inverted and cultured at 37°C overnight. Each plate was inoculated with a single colony in 5.0 mL of LB medium containing 50 μg / L Kana and cultured (37°C, 220 rpm, 6.0 h), and then sequenced. After sequencing, the correct mutant plasmid pET-28a(+)-OsUGT(C31M) was returned by the service company.

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

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

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

[0042] The remaining steps were the same as in (1), and the mutant plasmid pET-28a(+)-OsUGT(C31M / R310A) was obtained.

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

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

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

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

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

[0048] F354G_F: 5'-CGCATGCTGCTGTGGGCGCAGGTCTGACCCACTGTGGTT GGAAC-3'

[0049] F354G_R: 5'-GTTCCAACCACAGTGGGTCAGACCTGCGCCCACAGCAGC ATGCG-3'

[0050] The remaining steps are as in (1), and the mutant plasmid pET-28a(+)-OsUGT(C31M / R310A / Q342W / F354G) is obtained. 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-glucose transferase enzyme solution

[0052] The mutant plasmid pET-28a(+)-OsUGT(C31M / R310A / Q342W / F354G) is transformed into E. coli BL21(DE3) to obtain the recombinant engineering bacteria E. coli BL21(DE3)-OsUGT(C31M / R310A / Q342W / F354G). The recombinant engineering bacteria E. coli BL21(DE3)-OsUGT(C31M / R310A / Q342W / F354G) is inoculated into 4.0 mL of liquid LB medium at a ratio of 1%, and is cultured at 37°C with shaking (200 rpm) overnight. The overnight culture is inoculated into a large volume of liquid LB medium at a ratio of 1%, and is cultured at 37°C with shaking (200 rpm) until the OD600 value reaches 0.6-0.8. Then, 0.1 mM-1 mM IPTG is added, and the culture is incubated at 20-37°C with shaking for 12.0-16.0 h. After the induction is completed, the bacterial cells are collected by centrifugation, and the cells are resuspended with 50 mM phosphate buffer (pH 7.2). The cells are broken by ultrasonic treatment in an ice bath, and the broken solution is centrifuged. The supernatant is collected to obtain a crude enzyme solution containing the UDP-glucose transferase 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, 3.0 mg of AtSUS sucrose synthase, and 5.0 mg of the crude enzyme solution of the UDP-glucosyltransferase OsUGT (C31M / R310A / Q342W / F354G) mutant prepared in the third part, the total volume was 10.0 ml, and the pH was adjusted to 7.2. The reaction was carried out at 37°C and 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated. The enzyme activity assay result was 670.6 ± 2.6 U / mg.

[0055] 5. Reaction temperature assay

[0056] Using 2.0 g of rebaudioside A as a substrate, 0.7 g of sucrose, 1.0 mg of ADP, 3.0 mg of AtSUS sucrose synthase, and 5.0 mg of the crude enzyme solution of the UDP-glucosyltransferase OsUGT (C31M / R310A / Q342W / F354G) mutant prepared in the third part, the total volume was 10.0 ml, and the pH was adjusted to 7.2. The reaction was carried out at 37°C and 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated. The enzyme activity assay result was 670.6 ± 2.6 U / mg.

[0057] Temperature 5 min 15 min 30 min 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] Embodiment of the present application

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

[0060] 1. Preparation of parent plasmid

[0061] The desired gene fragment was synthesized according to the codon optimization of the amino acid sequence of UDP-glucosyltransferase derived from rice (Oryza sativa Japonica Group) (GenBank Accession No.: XP_015629141.1), and was connected into a pET28a vector with Nde I and Xho I as the enzyme digestion sites at both ends, to obtain a UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid. The obtained parent plasmid was transformed into E. coli DH5α, and after Kana screening, colonies were picked for sequencing to determine the nucleotide sequence of the cloned UDP-glucosyltransferase parent, which is shown as SEQ ID NO: 1, and the amino acid sequence thereof is shown as SEQ ID NO: 2.

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

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

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

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

[0066] After PCR, 1 μL Dpn I enzyme was added, mixed thoroughly, and then incubated at 37°C for 1.0 h to digest the template plasmid. Then 5 μL of the PCR reaction solution was added to DH5α competent cells, which were placed on ice for 30 min, heated in a water bath at 42°C for 60 s, immediately placed on ice for 3 min, and then added to 500 μL of LB medium without antibiotics in a clean bench, and cultured at 37°C and 220 rpm for 60 min. The bacterial solution was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining 100 μL of bacterial solution was resuspended with a syringe, and 100 μL of the bacterial solution was removed with a pipette and added to LB solid medium containing Kana. The medium was evenly coated with a sterile coating rod, and after the bacterial solution was absorbed, the plate was inverted and cultured at 37°C overnight. Each plate was inoculated with a single colony in 5.0 mL of LB medium containing 50 μg / L Kana, and cultured at 37°C and 220 rpm for 6.0 h. The sample was then sequenced, and the correct mutant plasmid pET-28a(+)-OsUGT(Q36W) was returned by the service company after sequencing.

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

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

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

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

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

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

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

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

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

[0076] F354G_F: 5'-CGCATGCTGCTGTGGGCGCAGGTCTGACCCACTGTGGTT GGAAC-3'

[0077] F354G_R: 5'-GTTCCAACCACAGTGGGTCAGACCTGCGCCCACAGCAGC ATGCG-3'

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

[0079] 3. Preparation of UDP-glucose transferase enzyme solution

[0080] The mutant plasmid pET-28a(+)-OsUGT(Q36W / E299K / R310A / F354G) is transformed into E. coli BL21(DE3) to obtain the recombinant engineering bacteria E. coli BL21(DE3)-OsUGT(Q36W / E299K / R310A / F354G). The recombinant engineering bacteria E. coli BL21(DE3)-OsUGT(Q36W / E299K / R310A / F354G) is inoculated into 4.0 mL of liquid LB medium at a ratio of 1%, and is cultured at 37°C with shaking (200 rpm) overnight. The overnight culture is inoculated into a large volume of liquid LB medium at a ratio of 1%, and is cultured at 37°C with shaking (200 rpm) until the OD600 value reaches 0.6-0.8. Then, 0.1 mM-1 mM IPTG is added, and the culture is incubated at 20-37°C with shaking for 12.0-16.0 h. After the induction is completed, the bacterial cells are collected by centrifugation, and the cells are resuspended with 50 mM phosphate buffer (pH 7.2). The cells are broken by ultrasonic wave in an ice bath, and the broken solution is centrifuged. The supernatant is collected to obtain the crude enzyme solution containing UDP-glucose transferase OsUGT(Q36W / E299K / R310A / F354G).

[0081] 4. ADP-dependent enzyme activity assay

[0082] Using 2.0 g of rebaudioside A as substrate, 0.7 g of sucrose, 1.0 mg of ADP, 3.0 mg of AtSUS sucrose synthase, and 5.0 mg of the crude enzyme solution of the UDP-glucosyltransferase OsUGT (Q36W / E299K / R310A / F354G) mutant prepared in the third part, the total volume was adjusted to 10.0 ml, and the pH was adjusted to 7.2. The reaction was carried out at 37°C and 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated. The enzyme activity assay result was 378.6 ± 1.9 U / mg.

[0083] 5. Reaction temperature assay

[0084] Using 2.0 g of rebaudioside A as substrate, 0.7 g of sucrose, 1.0 mg of ADP, 3.0 mg of AtSUS sucrose synthase, and 5.0 mg of the crude enzyme solution of the UDP-glucosyltransferase OsUGT (Q36W / E299K / R310A / F354G) mutant prepared in the third part, the total volume was adjusted to 10.0 ml, and the pH was adjusted to 7.2. The reaction was carried out at 37°C and 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated. The enzyme activity assay result was 378.6 ± 1.9 U / mg.

[0085] Temperature 5 min 15 min 30 min 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 parent plasmid

[0088] The desired gene fragment was synthesized according to the codon optimization of the amino acid sequence of UDP-glucosyltransferase derived from rice (Oryza sativa Japonica Group) (GenBank Accession No.: XP_015629141.1), and was connected into a pET28a vector, with Nde I and Xho I as the enzyme digestion sites at both ends, to obtain a UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid. The obtained parent plasmid was transformed into E. coli DH5α, and after Kana screening, colonies were picked for sequencing to determine the nucleotide sequence of the cloned UDP-glucosyltransferase parent, which is shown as SEQ ID NO: 1, and the amino acid sequence thereof is shown as SEQ ID NO: 2.

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

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

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

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

[0093] After PCR, 1 μL Dpn I enzyme was added, mixed thoroughly, and then incubated at 37°C for 1.0 h to digest the template plasmid. Then 5 μL of the PCR reaction solution was added to DH5a competent cells, which were placed on ice for 30 min, heated at 42°C for 60 s in a water bath, immediately placed on ice for 3 min, and then added to 500 μL of LB medium without antibiotics in a clean bench and cultured (37°C, 220 rpm, 60 min). The bacterial solution was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining 100 μL of bacterial solution was resuspended by blowing and sucking with a syringe, and 100 μL of the bacterial solution was removed with a pipette and added to LB solid medium containing Kana. The medium was evenly coated with a sterile coating rod, and after the bacterial solution was absorbed, the plate was inverted and cultured at 37°C overnight. Each plate was inoculated with a single colony in 5.0 mL of LB medium containing 50 μg / L Kana and cultured (37°C, 220 rpm, 6.0 h), and then sequenced. After sequencing, the correct mutant plasmid pET-28a(+)-OsUGT(K290E) was returned by the service company.

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

[0095] Q342W_F: '-GCAACCCGCTGGGTACCGTGGATGAGCATCCTGGCGCATGCTGCTG-3'

[0096] Q342W_R: '-CAGCAGCATGCGCCAGGATGCTCATCCACGGTACCCAGCGGGTTGC-3'

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

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

[0099] F354G_F: '-CGCATGCTGCTGTGGGCGCAGGTCTGACCCACTGTGGTTGGAAC-3'

[0100] F354G_R: '-GTTCCAACCACAGTGGGTCAGACCTGCGCCCACAGCAGCATGCG-3'

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

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

[0103] L367I_F: 5'-GGAACTCCACTATCGAAGGCATCATGTTCGGTCACCCTCT GATTATGCTGCCG-3'

[0104] L367I_R: 5'-CGGCAGCATAATCAGAGGGTGACCGAACATGATGCCTTC GATAGTGGAGTTCC-3'

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

[0106] 3. Preparation of UDP-glucose transferase enzyme solution

[0107] The mutant plasmid pET-28a(+)-OsUGT(K290E / Q342W / F354G / L367I) is transformed into E. coli BL21(DE3) to obtain recombinant engineering bacteria E. coli BL21(DE3)-OsUGT(K290E / Q342W / F354G / L367I). The recombinant engineering bacteria E. coli BL21(DE3)-OsUGT(K290E / Q342W / F354G / L367I) is inoculated into 4.0 mL of liquid LB medium at a ratio of 1%, and is cultured at 37°C with shaking (200 rpm) overnight. The overnight culture is inoculated into a large volume of liquid LB medium at a ratio of 1%, and is cultured at 37°C with shaking (200 rpm) until the OD600 value reaches 0.6-0.8. Then, 0.1 mM-1 mM IPTG is added, and the culture is incubated at 20-37°C with shaking for 12.0-16.0 h. After the induction is completed, the bacterial cells are collected by centrifugation, and the cells are resuspended in 50 mM phosphate buffer (pH 7.2). The cells are broken by ultrasonic treatment in an ice bath, and the broken solution is centrifuged. The supernatant is collected to obtain a crude enzyme solution containing UDP-glucose transferase OsUGT(K290E / Q342W / F354G / L367I).

[0108] 4. ADP-dependent enzyme activity assay

[0109] Using 2.0 g of rebaudioside A as substrate, 0.7 g of sucrose, 1 mg of ADP, 3.0 mg of AtSUS sucrose synthase, and 5.0 mg of the crude enzyme solution of the UDP-glucosyltransferase OsUGT (K290E / Q342W / F354G / L367I) mutant prepared in the third part, the total volume of each was 10.0 ml, and the pH was adjusted to 7.2. The reaction was carried out at 37°C and 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated. The enzyme activity assay result was 523.6 ± 3.2 U / mg.

[0110] 5. Reaction temperature assay

[0111] Using 2.0 g of rebaudioside A as substrate, 0.7 g of sucrose, 1.0 mg of ADP, 3.0 mg of AtSUS sucrose synthase, and 5.0 mg of the crude enzyme solution of the UDP-glucosyltransferase OsUGT (K290E / Q342W / F354G / L367I) mutant prepared in the third part, the total volume of each was 10.0 ml, and the pH was adjusted to 7.2 using 10 M sodium hydroxide. The temperature was 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C, and 50 μL of the reaction solution was taken at 5 min, 15 min, 30 min, or 1.0 h as a liquid sample for HPLC detection. The temperature was 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C, and the conversion rate of rebaudioside D in the reaction was as shown in the following table:

[0112] Temperature 5 min 15 min 30 min 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] ADP-dependent enzyme activity assay of the parent UDP-glucosyltransferase

[0114] 1. Preparation of the parent plasmid

[0115] According to the amino acid sequence of UDP-glucosyltransferase derived from rice (Oryza sativa Japonica Group) (GenBank Accession No: XP_015629141.1), a desired gene fragment was synthesized and connected into a pET28a vector, with Nde I and Xho I as the enzyme cutting sites at both ends, to obtain a UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid. The obtained parent plasmid was transformed into E. coli DH5α, and after Kana screening, colonies were picked for sequencing. The nucleotide sequence of the cloned UDP-glucosyltransferase parent was shown in SEQ ID NO: 1, and the amino acid sequence was shown in SEQ ID NO: 2.

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

[0117] The UDP-glucosyltransferase parent pET-28a(+)-OsUGT plasmid was transformed into E. coli BL21 (DE3) to obtain recombinant engineering bacteria E. coli BL21 (DE3)-OsUGT. The recombinant engineering bacteria E. coli BL21 (DE3)-OsUGT was inoculated into 4.0 mL liquid LB medium at a proportion of 1%, and cultured at 37°C with shaking (200 rpm) overnight. The overnight culture was inoculated into a large volume of liquid LB medium at a 1% inoculation amount, and cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. Then, 0.1 mM-1.0 mM IPTG was added, and the culture was incubated at 20-37°C with shaking for 12.0-16.0 h. After induction, the bacterial cells were collected by centrifugation, and the cells were resuspended with 50 mM phosphate buffer (pH 7.2). The cells were broken by ultrasonic wave in an ice bath, and the broken solution was centrifuged. The supernatant containing the UDP-glucosyltransferase parent was obtained.

[0118] 3. Determination of ADP-dependent enzyme activity

[0119] Rebaudioside A 2.0 g was used as the substrate, sucrose 0.7 g, ADP 1.0 mg, AtSUS sucrose synthase 3.0 mg, and 5.0 mg of the UDP-glucosyltransferase parent crude enzyme solution prepared in the second part was added, with a total volume of 10.0 mL. The pH was adjusted to 7.2, and the reaction was carried out at 37°C with 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated. The enzyme activity determination result was 247.67±2.3 U / mg.

[0120] 4. Determination of reaction temperature

[0121] Preparation of 8 parts of 2.0 g rebaudioside A, 0.7 mg sucrose, 1.0 mg ADP, 3.0 mg AtSUS sucrose synthase, and 5.0 mg of the crude enzyme solution of the UDP-glucosyltransferase parent prepared in Section 2, with a total volume of 10.0 ml per part, adjust with 10 M sodium hydroxide, adjust the pH to 7.2, and set the temperature to 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C, respectively. Take 50 μL of the reaction solution at 5 min, 15 min, 30 min, and 1.0 h, respectively, as the liquid sample for HPLC sample detection. The conversion rates of rebaudioside D at temperatures of 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C, respectively, are shown in the following table:

[0122] Temperature 5 min 15 min 30 min 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 by UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant

[0124] 1. Reaction pH optimization

[0125] Rebaudioside A 2.0 g, sucrose 0.7 g, ADP 1.0 mg, and 5.0 mg of the crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant and 3.0 mg of AtSUS sucrose synthase were stirred to completely dissolve, and the total volume was adjusted to 10.0 ml per part. The pH was adjusted to 6.0, 6.5, 7.0, and 7.5. The reaction was carried out at 60°C and 200 rpm for 1.0 h. After the reaction was completed, 50 μL of the reaction solution was taken, filtered through a 0.22 μm microfiltration membrane, and used as the liquid sample for HPLC sample detection. 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 reaction pH was 7.0.

[0126] 2. Reaction time optimization

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

[0128] 3. Substrate dosage optimization

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

[0130] 4. UDP-glucosyltransferase enzyme dosage optimization

[0131] Prepare 2.0g of rebaudioside A, 0.7g of sucrose, and 1.0mg of ADP, stirring until completely dissolved. Add 4.0mg, 6.0mg, 8.0mg, 10.0mg of the crude enzyme solution of the UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant, and 3.0mg of AtSUS sucrose synthase, respectively, to a total volume of 10.0ml. Adjust the pH to 7.2. Incubate the reaction at 60°C, 200rpm for 1.0h. After completion of the reaction, remove 50μL of the reaction solution, filter through a 0.22μm microfiltration membrane, and load it onto HPLC for analysis. When the amount of crude enzyme solution of 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 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 UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant was 8.0 mg, the conversion rate of rebaudioside D was 91.3%; when the amount of crude enzyme solution of UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant was 10.0 mg, the conversion rate of rebaudioside D was 91.3%. The amount of the crude enzyme solution of the optimal UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant was 8.0 mg.

[0132] Example 5 Preparation of Rebaudioside D by UDP-glucosyltransferase Q36W / E299K / R310A / F354G Mutant

[0133] 1. Reaction pH optimization

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

[0135] 2. Reaction time optimization

[0136] Rebaudioside A 2.0 g, sucrose 0.7 g, ADP 1.0 mg, stirred to completely dissolve, added 5.0 mg of crude enzyme solution of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant and 3.0 mg of AtSUS sucrose synthase, total volume of each portion was 10.0 ml, adjusted pH to 7.2. Reaction was carried out at 70°C, 200 rpm for 15 min, 30 min, 1.0 h, 1.5 h respectively, after the reaction, 50 μL of reaction solution was taken, filtered through 0.22 μm microfiltration membrane, as liquid sample for HPLC sample detection. When the reaction time was 15 min, the conversion rate of rebaudioside D was 28.6%; when the reaction time was 30 min, the conversion rate of rebaudioside D was 50.1%; when the reaction time was 1.0 h, the conversion rate of rebaudioside D was 68.3%; when the reaction time was 1.5 h, the conversion rate of rebaudioside D was 68.5%.

[0137] 3. Substrate dosage optimization

[0138] Rebaudioside A 1.6 g, rebaudioside A 2.0 g, rebaudioside A 2.4 g, rebaudioside A 3.0 g, sucrose 0.7 g, ADP 1.0 mg were respectively prepared, stirred to completely dissolve, added 5.0 mg of crude enzyme solution of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant and 3.0 mg of AtSUS sucrose synthase, total volume of each portion was 10.0 ml, adjusted pH to 7.2. Reaction was carried out at 70°C, 200 rpm for 1.0 h, after the reaction, 50 μL of reaction solution was taken, filtered through 0.22 μm microfiltration membrane, as liquid sample for HPLC sample detection. 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%; when rebaudioside A was 3.0 g, the conversion rate of rebaudioside D was 72.3%.

[0139] 4. UDP-glucosyltransferase enzyme dosage optimization

[0140] Reactions were carried out at 70°C, 200 rpm for 1.0 h. When the amount of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant crude enzyme solution was 4.0 mg, the conversion rate of rebaudioside D was 70.9%; when the amount of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant crude enzyme solution was 6.0 mg, the conversion rate of rebaudioside D was 73.4%; when the amount of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant crude enzyme solution was 8.0 mg, the conversion rate of rebaudioside D was 73.4%; when the amount of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant crude enzyme solution was 10.0 mg, the conversion rate of rebaudioside D was 73.4%.

[0141] Example 6 Preparation of rebaudioside D by UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant

[0142] 1. Reaction pH optimization

[0143] Reactions were carried out at 70°C, 200 rpm for 1.0 h. When the amount of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant crude enzyme solution was 4.0 mg, the conversion rate of rebaudioside D was 70.9%; when the amount of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant crude enzyme solution was 6.0 mg, the conversion rate of rebaudioside D was 73.4%; when the amount of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant crude enzyme solution was 8.0 mg, the conversion rate of rebaudioside D was 73.4%; when the amount of UDP-glucosyltransferase Q36W / E299K / R310A / F354G mutant crude enzyme solution was 10.0 mg, the conversion rate of rebaudioside D was 73.4%.

[0144] 2. Reaction time optimization

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

[0146] 3. Substrate dosage optimization

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

[0148] 4. UDP-glucosyltransferase enzyme dosage optimization

[0149] Rebaudioside A 2.0 g, sucrose 0.7 g, ADP 1 mg, and the crude enzyme solution of UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant and AtSUS sucrose synthase were added in an amount of 4.0 mg, 6.0 mg, 8.0 mg, 10.0 mg, and 3.0 mg, respectively, to a total volume of 10 ml, and the pH was adjusted to 7.2. The reaction was carried out at 65°C and 200 rpm for 1.0 h, and 50 μL of the reaction solution was taken after the reaction and filtered through a 0.22 μm microfiltration membrane as a liquid sample for HPLC detection. When the amount of the crude enzyme solution of UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant was 4.0 mg, the conversion rate of rebaudioside D was 66.4%; when the amount of the crude enzyme solution of UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant was 6.0 mg, the conversion rate of rebaudioside D was 68.5%; when the amount of the crude enzyme solution of UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant was 8.0 mg, the conversion rate of rebaudioside D was 68.8%; and when the amount of the crude enzyme solution of UDP-glucosyltransferase K290E / Q342W / F354G / L367I mutant was 10.0 mg, the conversion rate of rebaudioside D was 68.8%.

[0150] Industrial applicability

[0151] Rebaudioside A 6.0 kg, sucrose 2.1 g, ADP 2.5 g, and the crude enzyme solution of UDP-glucosyltransferase C31M / R310A / Q342W / F354G mutant and AtSUS sucrose synthase were added in an amount of 13.5 g and 8.1 g, respectively, to a total volume of 30.0 L, and the pH was adjusted to 7.0. The reaction was carried out at 60°C, and 50 μL of the reaction solution was taken after the reaction and filtered through a 0.22 μm microfiltration membrane as a liquid sample for HPLC detection. After 2 h of reaction, 90.1% conversion was achieved.

[0152] Free content of sequence listing

[0153] SEQ ID NO. 1: Nucleotide sequence of UDP-glucosyltransferase of Oryza sativa Japonica Group.

[0154] SEQ ID NO. 2: Amino acid sequence of UDP-glucosyltransferase of Oryza sativa Japonica Group.

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

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

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

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

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

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

Claims

1. A UDP-glucosyltransferase mutant, characterized in that The UDP-glucose transferase mutant catalyzes rebaudioside A, sucrose and ADP to produce rebaudioside D.

2. A UDP-glucosyltransferase mutant according to claim 1, characterized in that The UDP-glucosyltransferase mutant is mutated from the parent as shown in SEQ ID NO: 2 to C31M / R310A / Q342W / F354G or Q36W / E299K / R310A / F354G or K290E / Q342W / F354G / L367I.

3. A method for preparing rebaudioside D using a UDP-glucosyltransferase mutant, characterized in that: Rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant according to claim 2 are used as substrates to catalyze the reaction to produce rebaudioside D.

4. The method for preparing rebaudioside D using a UDP-glucosyltransferase mutant according to claim 3, wherein: Rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant according to claim 2 are used as substrates, the pH is 6.0-7.5, the temperature is 35-70° C., and the reaction time is 15 min-1.5 h to catalyze the reaction to produce rebaudioside D.

5. The method for preparing rebaudioside D using a UDP-glucosyltransferase mutant according to claim 4, wherein: Rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant according to claim 2 are used as substrates, the pH is 7.0, the temperature is 60-70° C., and the reaction time is 1.5 h to catalyze the reaction to produce rebaudioside D.

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

3.

7. A method for preparing rebaudioside D using a UDP-glucosyltransferase mutant, characterized in that: Rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant according to claim 6 are used as substrates to catalyze the reaction to produce rebaudioside D.

8. The method for preparing rebaudioside D using a UDP-glucosyltransferase mutant according to claim 7, wherein: Rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant according to claim 6 are used as substrates, the pH is 6.0-7.5, the temperature is 35-70° C., and the reaction time is 15 min-1.5 h to catalyze the reaction to produce rebaudioside D.

9. The method for preparing rebaudioside D using a UDP-glucosyltransferase mutant according to claim 8, wherein: Rebaudioside A, sucrose, ADP, AtSUS sucrose synthase and the UDP-glucosyltransferase mutant according to claim 6 are used as substrates, the pH is 7.0, the temperature is 60° C., and the reaction time is 1.5 h to catalyze the reaction to produce rebaudioside D.

10. A method for preparing rebaudioside D using a UDP-glucosyltransferase mutant, characterized in that: 6.0 kg of rebaudioside A, 2.1 kg of sucrose, 2.5 g of ADP, a total volume of 30.0 L, 13.5 g of the crude enzyme solution of the UDP-glucosyltransferase mutant described in claim 6, and 8.1 g of AtSUS sucrose synthase were added. The pH was adjusted to 7.

0. The reaction was carried out at 60°C to catalyze the production of rebaudioside D.