Sucrose synthase mutant and method for preparing rd using same

Abstract

Disclosed in the present invention are a sucrose synthase mutant and a method for preparing RD using same. The sucrose synthase mutant is based on the amino acid sequence shown in SEQ ID NO: 2 and contains a mutation in at least one of the following sites: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R. By means of site-directed mutagenesis of sucrose synthase, the enzyme activity of the sucrose synthase mutant is improved compared with that of the wild type. Also, some mutants can still maintain a high conversion rate in a wide pH range.

Description

Sucrose synthase mutant and method for preparing RD therefrom TECHNICAL FIELD

[0001] The present application relates to the technical field of steviol glycoside production, and in particular to a sucrose synthase mutant and a method for preparing RD therefrom. BACKGROUND

[0002] Stevioside is a natural glycoside sweetener extracted from the leaves of the plant Stevia rebaudiana (Bertoni) H. de Buncheri. The chemical formula of steviol glycoside is C 38 H 60 O 18 , the CAS number is 57817-89-7, and it has the characteristics of low heat (heat value is only 1 / 300 of sucrose), high sweetness (about 300 times of sucrose) and strong stability. International sweetener industry data shows that steviol glycoside has been widely used in the production of food, beverage and seasoning in Asia, North America, South America and European countries.

[0003] RD (Reb D, also known as rebaudioside D) is a natural sweetener with high sweetness and low heat. It stands out among many natural sweeteners due to its higher sweetness, less bitter aftertaste and similar taste to sucrose. In the early stage, RD was mainly extracted from the plant Stevia rebaudiana, but its proportion in Stevia rebaudiana was less than 1%. This method is costly and difficult to meet market demand. Currently, the main synthesis methods of RD include chemical method, fermentation method and biological enzyme method. Biological catalysis method can obtain high concentration of RD, reduce environmental pollution and improve the yield of target product RD.

[0004] Sucrose synthase (SuSy) is an enzyme that exists in plants and some microorganisms. It plays a key role in sucrose metabolism and is mainly responsible for catalyzing sucrose synthesis and decomposition reactions. It can catalyze the reaction of ADP to ADPG, which is not only the core of carbohydrate metabolism, but also indirectly provides the material basis for the conversion of rebaudioside A (Reb A) to rebaudioside D (Reb D) by providing a key sugar donor. The activity of SuSy directly determines the concentration level of ADPG in the system, and the availability of ADPG is a key factor limiting the glycosylation efficiency of Reb A.

[0005] Sucrose synthase can accelerate the rate and conversion of rebaudioside A to rebaudioside D by catalyzing ADP to ADPG. However, the catalytic activity of wild-type sucrose synthase is not high, the affinity for ADP and the efficiency of ADPG generation are limited, and its catalytic direction is more inclined to sucrose synthesis, resulting in insufficient ADPG supply and limiting the efficient synthesis of Reb D. Therefore, it is necessary to mutate the wild-type sucrose synthase by genetic modification technology to further improve the activity of sucrose synthase, provide sufficient substrate for the glycosylation reaction of Reb A to Reb D, and thus improve the synthesis efficiency of Reb D. SUMMARY

[0006] To solve the above technical problems, the present application further improves the activity of sucrose synthase by a mutant of sucrose synthase, solves the problem of low catalytic efficiency of wild-type sucrose synthase, improves the conversion efficiency of RA to RD, and realizes the efficient, energy-saving and large-scale production of rebaudioside D.

[0007] To achieve the above purpose, the present application adopts the following technical solutions:

[0008] The sucrose synthase mutant can regenerate ADP-glucose from sucrose and ADP to catalyze the conversion of rebaudioside A to rebaudioside D in cooperation with β-1, 2-glucosidase; the sucrose synthase mutant contains at least one mutation of D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R based on the amino acid sequence shown in SEQ ID NO: 2.

[0009] As a preferred technical solution, the amino acid sequence of the sucrose synthase mutant comprises one of the following mutation combinations based on the wild-type sucrose synthase shown in SEQ ID NO: 2:

[0010] R567T, R636Q and A642N;

[0011] G514L, R567T, A642N and T742R;

[0012] G514L, R567T, A642N, L667T and T742R;

[0013] D296Q, H531L, D569Q, E610N and E663N.

[0014] As a preferred technical solution, the nucleotide sequence of the sucrose synthase mutant is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0015] As a preferred technical solution, the beta-1, 2-glucosidase is a UDP-glucosyltransferase mutant.

[0016] A method for preparing rebaudioside D by using a sucrose synthase mutant, wherein the sucrose synthase mutant and the beta-1, 2-glucosidase are used as catalysts, and rebaudioside A, sucrose and ADP are used as substrates to generate rebaudioside D.

[0017] As a preferred technical solution, the reaction temperature in the catalytic reaction is controlled at 55-80℃, the pH is 4.0-8.0, and the reaction time is 0.5-18h.

[0018] As a preferred technical solution, the reaction temperature in the catalytic reaction is controlled at 55-75℃, the pH is 5.0-7.0, and the reaction time is 4-18h.

[0019] As a preferred technical solution, the reaction temperature in the catalytic reaction is controlled at 65-70℃, the pH is 6.0, and the reaction time is 6h.

[0020] As a preferred technical solution, the reaction is carried out in an aqueous solution containing the following components: the initial reaction concentration of rebaudioside A is 160-300g / L, the initial reaction concentration of sucrose is 60-80g / L, the initial reaction concentration of ADP is 0.4-4.4g / L, the initial reaction concentration of beta-1, 2-glucosidase is 0.4-0.6g / L, the total protein concentration of the crude enzyme solution of the NeSUS sucrose synthase mutant according to any one of claims 1-4 in the reaction system is 0.3-0.5g / L, and the balance is solvent.

[0021] As a preferred technical solution, the reaction is carried out in an aqueous solution containing the following components: the initial reaction concentration of rebaudioside A is 200g / L, the initial reaction concentration of sucrose is 70g / L, the initial reaction concentration of ADP is 0.7g / L, the initial reaction concentration of beta-1, 2-glucosidase is 0.5g / L, the total protein concentration of the crude enzyme solution of the NeSUS sucrose synthase mutant according to any one of claims 1-4 in the reaction system is 0.5g / L, and the balance is pure water; the pH is adjusted to 6.0, the reaction is carried out at 60℃ for 18h, and rebaudioside D is generated by catalytic reaction. Beneficial effects

[0022] The sucrose synthase mutant in the present application can regenerate ADP-glucose by using sucrose and ADP, so as to catalyze the conversion of rebaudioside A to rebaudioside D in cooperation with the beta-1, 2-glucosidase.

[0023] Specifically, the sucrose synthase mutant enzyme activity of the present application is increased by 12% to 38% compared with the wild type (see the test results of the sucrose synthase mutant and its parent enzyme activity in the comparative examples), the conversion efficiency is significantly improved, and the adaptability to reaction conditions is stronger, and part of the mutants still maintains a high conversion rate in a wider pH range. The characteristics of the mutant enhance its applicability and feasibility in industrial production, and the impact of reaction condition fluctuations on the reaction results. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is an HPLC spectrum of the reaction catalyzed by the sucrose synthase NeSUS(G514L / R567T / A642N / L667T / T742R) of Example 3 of the present application to synthesize rebaudioside D (reaction 0 hours);

[0025] Figure 2 is an HPLC spectrum of the reaction catalyzed by the sucrose synthase NeSUS(G514L / R567T / A642N / L667T / T742R) of Example 3 of the present application to synthesize rebaudioside D (reaction 1 hour);

[0026] Figure 3 is an HPLC spectrum of the reaction catalyzed by the sucrose synthase NeSUS(G514L / R567T / A642N / L667T / T742R) of Example 3 of the present application to synthesize rebaudioside D (reaction 2 hours);

[0027] Figure 4 is an HPLC spectrum of the reaction catalyzed by the sucrose synthase NeSUS(G514L / R567T / A642N / L667T / T742R) of Example 3 of the present application to synthesize rebaudioside D (reaction 18 hours). DETAILED DESCRIPTION

[0028] In order to make the present application easy to understand, the present application will be described in detail below in combination with specific embodiments. However, before the detailed description of the present application, it should be understood that the present 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 intended to be limiting.

[0029] 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 be used in the practice of the present application, the preferred methods and materials are now described.

[0030] TB medium: yeast powder 23.6 g / L, tryptone 11.8 g / L, potassium phosphate dibasic 9.4 g / L, potassium phosphate monobasic 2.2 g / L, glycerol 4.0 mL / L.

[0031] LB medium: tryptone 10.0 g / L, sodium chloride 10.0 g / L, yeast extract 5.0 g / L.

[0032] PCR reaction system: 10x high-fidelity buffer containing MgSO4 5 μL, 10 mM dNTP mixture 1 μL, upstream primer solution (10 mM) 1 μL, downstream primer solution (10 mM) 1 μL, template plasmid solution 1 μL, high-fidelity DNA polymerase, 2.5 U / μl 1.2 μL, ddH2O 39.8 μL.

[0033] PCR setting 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, and incubation at 12℃.

[0034] Example 1 Sucrose synthase (R567T / R636Q / A642N) mutation

[0035] 1. Preparation of sucrose synthase parent plasmid

[0036] The sucrose synthase NeSUS (Genbank No. CAD85125.1) derived from Nitrosomonas europaea was codon-optimized to synthesize the required gene fragment, which was connected to the pET28a vector, and the two end enzyme digestion sites were Nde I and Xho I, respectively, to obtain the sucrose synthase parent pET-28a(+)-NeSUS plasmid. The obtained parent plasmid was transformed into Escherichia coli DH5α, and after Kana screening, the colonies were picked for sequencing to determine the nucleotide sequence of the cloned sucrose synthase parent, which is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0037] 2. Site-directed mutation of sucrose synthase

[0038] (1) The wild-type sucrose synthase (amino acid sequence as shown in SEQ ID NO. 1) was mutated by the method of whole plasmid site-directed mutation PCR. The sucrose synthase parent pET-28a(+)-NeSUS plasmid was used as the template, and R567T_F / R567T_R was used as the primer pair for the first round of site-directed mutation PCR. The primers were designed as follows:

[0039] R567T_F: 5'-GCTGATTTTTACCATGGCAactCTGGATCGCATTAAGAAC-3'

[0040] R567T_R: 5'-GTTCTTAATGCGATCCAGagtTGCCATGGTAAAAATCAGC-3'

[0041] 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 (37°C, 220 rpm, 60 min). The bacterial solution was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining bacterial solution was resuspended by blowing and sucking with a gun head. Then 100 μL of the bacterial solution was removed with a pipette gun and added to LB solid medium containing Kana, which was evenly coated with a sterile coating rod. After the bacterial solution was absorbed, the plate was inverted and cultured overnight at 37°C. Each plate was inoculated with a single colony in 5.0 mL of LB medium containing 50 μg / mL Kana and cultured (37°C, 220 rpm, 6.0 h) before sequencing. After sequencing, the correct mutant plasmid pET-28a(+)-NeSUS(R567T) was returned by the service company.

[0042] (2) The pET-28a(+)-NeSUS(R567T) was used as the template plasmid, and R636Q_F / R636Q_R was used as the primer pair for the second round of site-directed mutagenesis. The primers were designed as follows:

[0043] R636Q_F: 5'-AGGTTCGCTGGCTGGGTATGcaaCTGGACAAAAACCTGGCAG-3'

[0044] R636Q_R: 5'-CTGCCAGGTTTTTGTCCAGttgCATACCCAGCCAGCGAACCT-3'

[0045] The remaining steps were as in (1), and the mutant plasmid pET-28a(+)-NeSUS(R567T / R636Q) was obtained.

[0046] (3) The pET-28a(+)-NeSUS(R567T / R636Q) was used as the template plasmid, and A642N_F / A642N_R was used as the primer pair for the third round of site-directed mutagenesis. The primers were designed as follows:

[0047] A642N_F: 5'-CTGGACAAAAACCTGaatGGCGAACTGTATCGC-3'

[0048] A642N_R: 5'-GCGATACAGTTCGCCattCAGGTTTTTGTCCAG-3'

[0049] The remaining steps are as in (1), and a mutant plasmid pET-28a(+)-NeSUS(R567T / R636Q / A642N) is obtained. The nucleotide sequence of the mutant sucrose synthase (R567T / R636Q / A642N) after mutation is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

[0050] 3. Preparation of sucrose synthase (R567T / R636Q / A642N) mutant enzyme solution

[0051] The mutant plasmid pET-28a(+)-NeSUS(R567T / R636Q / A642N) is transformed into E. coli BL21(DE3) to obtain a recombinant engineering bacterium E. coli BL21(DE3)-NeSUS(R567T / R636Q / A642N). A single colony of the recombinant engineering bacterium E. coli BL21(DE3)-NeSUS(R567T / R636Q / A642N) is inoculated into 4.0 mL of Kana-resistant LB liquid medium, and cultured at 37°C with shaking (200 rpm) overnight. The overnight culture is inoculated into 50 mL of Kana-resistant LB liquid medium at a 1% inoculation amount, and cultured at 37°C with shaking (200 rpm) until the OD 600 The value reaches 0.6-0.8, and 0.1 mM-1 mM IPTG is added at a final concentration to culture at 20-37°C with shaking for 12.0-16.0 h. After induction, 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 containing the sucrose synthase NeSUS(R567T / R636Q / A642N) mutant is obtained.

[0052] 4. Enzyme activity determination of sucrose synthase NeSUS(R567T / R636Q / A642N) mutant

[0053] With sucrose 26 mg and ADP 21 mg as substrates, 0.5 mg of the sucrose synthase NeSUS(R567T / R636Q / A642N) mutant crude enzyme solution prepared in the third part is added, the total volume is 1 mL, the pH is adjusted to 5.0, and the reaction is carried out at 75°C with 200 rpm for 30 min. Then, the sample is taken for HPLC detection, and the enzyme activity is calculated.

[0054] 5. Optimization of conversion rate of sucrose synthase NeSUS(R567T / R636Q / A642N) mutant at different reaction pH in RD

[0055] Take Rebaudioside A 2.0 g as the substrate, sucrose 0.7 g, ADP 44.0 mg, UDP-glucose transferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No: XP_015629141.1) 5.0 mg, and sucrose synthase NeSUS (R567T / R636Q / A642N) mutant crude enzyme liquid prepared in the third part 5.0 mg, respectively, and the total volume is 10.0 ml, and the reaction is carried out at 75°C, 200 rpm. After the reaction, the reaction solution is diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and then loaded on HPLC for detection to calculate the conversion rate.

[0056] At a temperature of 75°C, the pH gradient is set to 4.0, 5.0, 6.0, 7.0, and 8.0, and the conversion rate at different pH values is determined, and the results are as follows:

[0057] Example 2 Sucrose synthase (G514L / R567T / A642N / T742R) mutation

[0058] 1. Preparation of sucrose synthase parent plasmid

[0059] The sucrose synthase NeSUS (Genbank No: CAD85125.1) derived from Nitrosomonas europaea is codon-optimized to synthesize the required gene fragment, which is connected to the pET28a vector, and the two end enzyme digestion sites are Nde I and Xho I, respectively, to obtain the sucrose synthase parent pET-28a(+)-NeSUS plasmid. The obtained parent plasmid is transformed into E. coli DH5α, and after Kana screening, the colonies are picked for sequencing to determine the nucleotide sequence of the cloned sucrose synthase parent, which is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0060] 2. Site-directed mutation of sucrose synthase

[0061] (1) The wild-type sucrose synthase (amino acid sequence as shown in SEQ ID NO. 1) is mutated by the method of whole plasmid site-directed mutation PCR, and the sucrose synthase parent pET-28a(+)-NeSUS plasmid is used as the template, and G514L_F / G514L_R is used as the primer pair for the first round of site-directed mutation PCR, and the primers are designed as follows:

[0062] G514L_F: 5'-CAACATCGTATCCCCGcttGCTAACGCAGACATTTATTTTC-3'

[0063] G514L_R: 5'-GAAAATAAATGTCTGCGTTAGCaagCGGGGATACGATGTTG-3'

[0064] 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 the DH5a 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 (37°C, 220 rpm, 60 min). The bacterial solution was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining bacterial solution was resuspended by blowing and sucking with a gun head. Then 100 μL of the bacterial solution was removed with a pipette gun and added to LB solid medium containing Kana, which was evenly coated with a sterile coating rod. After the bacterial solution was absorbed, the plate was inverted and cultured overnight at 37°C. Each plate was inoculated with a single colony in 5.0 mL of LB medium containing 50 μg / mL Kana and cultured (37°C, 220 rpm, 6.0 h) before sequencing. After sequencing, the correct mutant plasmid pET-28a(+)-NeSUS(G514L) was returned by the service company.

[0065] (2) The second round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(G514L) as the template plasmid and R567T_F / R567T_R as the primer pair. The primers were designed as follows:

[0066] R567T_F: 5'-GCTGATTTTTACCATGGCAactCTGGATCGCATTAAGAAC-3'

[0067] R567T_R: 5'-GTTCTTAATGCGATCCAGagtTGCCATGGTAAAAATCAGC-3'

[0068] The remaining steps were as in (1), and the mutant plasmid pET-28a(+)-NeSUS(G514L / R567T) was obtained.

[0069] (3) The third round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(G514L / R567T) as the template plasmid and A642N_F / A642N_R as the primer pair. The primers were designed as follows:

[0070] A642N_F: 5'-CTGGACAAAAACCTGaatGGCGAACTGTATCGC-3'

[0071] A642N_R: 5'-GCGATACAGTTCGCCattCAGGTTTTTGTCCAG-3'

[0072] The remaining steps were as in (1), and mutant plasmid pET-28a(+)-NeSUS(G514L / R567T / A642N) was obtained.

[0073] (4) The fourth round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(G514L / R567T / A642N) as the template plasmid and T742R_F / T742R_R as the primer pair, and the primers were designed as follows:

[0074] T742R_F: 5'-GTGTAGCGTCTCGTTACagaTGGAAGCTGTATGCCGAAC-3'

[0075] T742R_R: 5'-GTTCGGCATACAGCTTCCAtctGTAACGAGACGCTACAC-3'

[0076] The remaining steps were as in (1), and mutant plasmid pET-28a(+)-NeSUS(G514L / R567T / A642N / T742R) was obtained. The nucleotide sequence of the mutant sucrose synthase (G514L / R567T / A642N / T742R) after mutation is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

[0077] 3. Preparation of sucrose synthase (G514L / R567T / A642N / T742R) mutant enzyme solution

[0078] The mutant plasmid pET-28a(+)-NeSUS(G514L / R567T / A642N / T742R) was transformed into E. coli BL21(DE3) to obtain the recombinant engineering bacteria E. coli BL21(DE3)-NeSUS(G514L / R567T / A642N / T742R). The recombinant engineering bacteria E. coli BL21(DE3)-NeSUS(G514L / R567T / A642N / T742R) was inoculated into 4.0 mL of LB liquid medium containing Kana resistance, and was cultured at 37°C with shaking (200 rpm) overnight. The overnight culture was inoculated into 50 mL of LB liquid medium containing Kana resistance at a 1% inoculation amount, and was cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. Then, 0.1 mM-1 mM IPTG was added to the medium, and the medium was cultured at 20-37°C with shaking for 12.0-16.0 h. After the induction, the bacteria were collected by centrifugation, and the cells were resuspended with 50 mM phosphate buffer (pH 7.2). The cells were broken by ultrasonic treatment in an ice bath, and the broken cells were centrifuged. The supernatant was collected to obtain the crude enzyme solution containing the sucrose synthase NeSUS(G514L / R567T / A642N / T742R) mutant.

[0079] 4. Enzyme activity determination of the sucrose synthase NeSUS(G514L / R567T / A642N / T742R) mutant

[0080] The sucrose 26 mg and ADP 21 mg were used as substrates, 0.5 mg of the crude enzyme solution of the sucrose synthase NeSUS(G514L / R567T / A642N / T742R) mutant prepared in the third part was added, the total volume was 1 mL, the pH was adjusted to 5.0, and the reaction was carried out at 75°C with shaking at 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated.

[0081] 5. Optimization of the conversion rate of the sucrose synthase NeSUS(G514L / R567T / A642N / T742R) mutant prepared in the third part under different reaction pHs in the RD

[0082] Take Rebaudioside A 2.0 g as the substrate, sucrose 0.7 g, ADP 44.0 mg, UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No: XP_015629141.1) 5.0 mg, and sucrose synthase NeSUS (G514L / R567T / A642N / T742R) mutant crude enzyme liquid prepared in the third part 5.0 mg, respectively, and the total volume is 10.0 ml, and the reaction is carried out at 75°C, 200 rpm. After the reaction, the reaction solution is diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and then loaded on HPLC for detection to calculate the conversion rate.

[0083] At a temperature of 75°C, the pH gradient is set to 4.0, 5.0, 6.0, 7.0, and 8.0, and the conversion rate at different pH values is determined, and the results are as follows:

[0084] Example 3 Sucrose synthase (G514L / R567T / A642N / L667T / T742R) mutant

[0085] 1. Preparation of sucrose synthase parent plasmid

[0086] The sucrose synthase NeSUS (Genbank No: CAD85125.1) derived from Nitrosomonas europaea is codon-optimized to synthesize the required gene fragment, which is connected to the pET28a vector, and the two end enzyme digestion sites are Nde I and Xho I, respectively, to obtain the sucrose synthase parent pET-28a(+)-NeSUS plasmid. The obtained parent plasmid is transformed into E. coli DH5α, and after Kana screening, the colonies are picked for sequencing to determine the nucleotide sequence of the cloned sucrose synthase parent, which is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0087] 2. Site-directed mutagenesis of sucrose synthase

[0088] (1) The wild-type sucrose synthase (amino acid sequence as shown in SEQ ID NO. 1) is mutated by the method of whole plasmid site-directed mutagenesis PCR, and the sucrose synthase parent pET-28a(+)-NeSUS plasmid is used as the template, and G514L_F / G514L_R is used as the primer pair for the first round of site-directed mutagenesis PCR, and the primers are designed as follows:

[0089] G514L_F: 5'-CAACATCGTATCCCCGcttGCTAACGCAGACATTTATTTTC-3'

[0090] G514L_R: 5'-GAAAATAAATGTCTGCGTTAGCaagCGGGGATACGATGTTG-3'

[0091] 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 the DH5a 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 (37°C, 220 rpm, 60 min). The bacterial solution was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining bacterial solution was resuspended by blowing and sucking with a gun head. Then 100 μL of the bacterial solution was removed with a pipette gun and added to LB solid medium containing Kana, which was evenly coated with a sterile coating rod. After the bacterial solution was absorbed, the plate was inverted and cultured overnight at 37°C. Each plate was inoculated with a single colony in 5.0 mL of LB medium containing 50 μg / mL Kana and cultured (37°C, 220 rpm, 6.0 h) before sequencing. After sequencing, the correct mutant plasmid pET-28a(+)-NeSUS(G514L) was returned by the service company.

[0092] (2) The second round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(G514L) as the template plasmid and R567T_F / R567T_R as the primer pair. The primers were designed as follows:

[0093] R567T_F: 5'-GCTGATTTTTACCATGGCAactCTGGATCGCATTAAGAAC-3'

[0094] R567T_R: 5'-GTTCTTAATGCGATCCAGagtTGCCATGGTAAAAATCAGC-3'

[0095] The remaining steps were as in (1), and the mutant plasmid pET-28a(+)-NeSUS(G514L / R567T) was obtained.

[0096] (3) The third round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(G514L / R567T) as the template plasmid and A642N_F / A642N_R as the primer pair. The primers were designed as follows:

[0097] A642N_F: 5'-CTGGACAAAAACCTGaatGGCGAACTGTATCGC-3'

[0098] A642N_R: 5'-GCGATACAGTTCGCCattCAGGTTTTTGTCCAG-3'

[0099] The remaining steps are as in (1), and the mutant plasmid pET-28a(+)-NeSUS(G514L / R567T / A642N) is obtained.

[0100] (4) The fourth round of site-directed mutagenesis is performed using pET-28a(+)-NeSUS(G514L / R567T / A642N) as the template plasmid and the primer pair L667T_F / L667T_R, and the primers are designed as follows:

[0101] L667T_F: 5'-GTTTGAAGCATTTGGTactACCATCATTGAGGCTATGG-3'

[0102] L667T_R: 5'-CCATAGCCTCAATGATGGTagtACCAAATGCTTCAAAC-3'

[0103] The remaining steps are as in (1), and the mutant plasmid pET-28a(+)-NeSUS(G514L / R567T / A642N / L667T) is obtained.

[0104] (5) The fifth round of site-directed mutagenesis is performed using pET-28a(+)-NeSUS(G514L / R567T / A642N / L667T) as the template plasmid and the primer pair T742R_F / T742R_R, and the primers are designed as follows:

[0105] T742R_F: 5'-GTGTAGCGTCTCGTTACagaTGGAAGCTGTATGCCGAAC-3'

[0106] T742R_R: 5'-GTGTAGCGTCTCGTTACagaTGGAAGCTGTATGCCGAAC-3'

[0107] The remaining steps are as in (1), and the mutant plasmid pET-28a(+)-NeSUS(G514L / R567T / A642N / L667T / T742R) is obtained. The nucleotide sequence of the mutant sucrose synthase (G514L / R567T / A642N / L667T / T742R) after mutagenesis is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

[0108] 3. Preparation of sucrose synthase (G514L / R567T / A642N / L667T / T742R) mutant enzyme solution

[0109] The mutant plasmid pET-28a(+)-NeSUS(G514L / R567T / A642N / L667T / T742R) was transformed into E. coli BL21(DE3) to obtain the recombinant engineering bacteria E. coli BL21(DE3)-NeSUS(G514L / R567T / A642N / L667T / T742R). The recombinant engineering bacteria E. coli BL21(DE3)-NeSUS(G514L / R567T / A642N / L667T / T742R) was inoculated into 4.0 mL of LB liquid medium containing Kana resistance, and cultured at 37°C with shaking (200 rpm) overnight. The overnight culture was inoculated into 50 mL of LB liquid medium containing Kana resistance 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 mM IPTG was added to the culture at a final concentration, and the culture was incubated at 20-37°C with shaking for 12.0-16.0 h. After the induction was completed, 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 was collected to obtain the crude enzyme solution containing the sucrose synthase NeSUS(G514L / R567T / A642N / L667T / T742R) mutant.

[0110] 4. Enzyme activity determination of the sucrose synthase NeSUS(G514L / R567T / A642N / L667T / T742R) mutant

[0111] Using sucrose 26 mg and ADP 21 mg as substrates, 0.5 mg of the crude enzyme solution of the sucrose synthase NeSUS(G514L / R567T / A642N / L667T / T742R) mutant prepared in the third part was added, the total volume was 1 mL, the pH was adjusted to 5.0, and the reaction was carried out at 75°C with shaking at 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated.

[0112] 5. Optimization of conversion rate of the sucrose synthase NeSUS(G514L / R567T / A642N / L667T / T742R) mutant prepared in the third part under different reaction pH in the preparation RD

[0113] Reactions were carried out at 75°C, 200 rpm, with 2.0 g of rebaudioside A as the substrate, 0.7 g of sucrose, 44.0 mg of ADP, 5.0 mg of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No: XP_015629141.1), and 5.0 mg of the crude enzyme solution of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant prepared in the third part, with a total volume of 10.0 ml. After the reaction was completed, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid sample for HPLC loading detection to calculate the conversion rate.

[0114] The conversion rates at different pH values were determined under the condition of a temperature of 75°C, with a pH gradient of 4.0, 5.0, 6.0, 7.0, and 8.0, and the results are shown in the following table:

[0115] 6, Conversion rate optimization of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant at different reaction temperatures under the optimal pH (pH 6.0) in RD

[0116] Reactions were carried out at 200 rpm, with 2.0 g of rebaudioside A as the substrate, 0.7 g of sucrose, 44.0 mg of ADP, 5.0 mg of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No: XP_015629141.1), and 3.0 mg of the crude enzyme solution of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant prepared in the third part, with a total volume of 10.0 ml. After the reaction was completed, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid sample for HPLC loading detection to calculate the conversion rate.

[0117] The conversion rates at different temperatures were determined under the condition of a pH of 6.0, with a temperature gradient of 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C, and the results are shown in the following table:

[0118] 7, ADP substrate feeding amount optimization of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant

[0119] ADP 44mg, ADP 18mg, ADP 8mg, ADP 4mg, sucrose 0.7g, RA 2g, stirring to completely dissolved, adding 5.0mg of UDP-glucose transferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No: XP_015629141.1) crude enzyme solution and 5.0mg sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant, each total volume of 10.0ml, adjust pH to 6. Reaction was carried out at 60℃, 200rpm for 6h respectively, after the reaction, the reaction solution was diluted 100 times with methanol, and filtered through 0.22μm microfiltration membrane as liquid sample for HPLC sample detection.

[0120] When ADP 44mg, the conversion rate of rebaudioside D was 87.1%; when ADP 18mg, the conversion rate of rebaudioside D was 85.6%; when ADP 8mg, the conversion rate of rebaudioside D was 84.2%; when ADP 4mg, the conversion rate of rebaudioside D was 77.6%.

[0121] 8、Sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant dosage optimization

[0122] Rebaudioside A 2.0g, sucrose 0.7g, ADP 8mg, stirring to completely dissolved, adding 5.0mg of UDP-glucose transferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No: XP_015629141.1) crude enzyme solution and sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant in an amount of 3.0mg, 4.0mg, 5.0mg respectively, each total volume of 10.0ml, adjust pH to 6. Reaction was carried out at 60℃, 200rpm for 18h respectively, after the reaction, the reaction solution was diluted 100 times with methanol, and filtered through 0.22μm microfiltration membrane as liquid sample for HPLC sample detection.

[0123] When the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant is used in an amount of 3.0 mg, the conversion rate of rebaudioside D is 84.6%; when the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant is used in an amount of 4.0 mg, the conversion rate of rebaudioside D is 82.3%; and when the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant is used in an amount of 5.0 mg, the conversion rate of rebaudioside D is 81%.

[0124] 9. Scale-up experiment of sucrose synthase mutant for preparing rebaudioside D

[0125] Rebaudioside A 6.0 kg, sucrose 2.1 kg, ADP 2.4 g, UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No. XP_015629141.1) 15.0 g, and a total volume of 30.0 L, are stirred to completely dissolve, 9 g of crude enzyme solution of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant is added, and the pH is adjusted to 6.0. The reaction is carried out at 60°C, and after the reaction is completed, the reaction solution is diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid sample for HPLC sample detection. After 18 h of reaction, the conversion rate is 82.1%.

[0126] From the 10 mL small test in the examples to the 30 L scale-up experiment, the rebaudioside A feed amount is increased from 2.0 g to 6.0 kg, and the conversion rate is decreased from 84.2% to 82.1%, with a decrease of only 2.1%, which indicates that the process has high stability. At the same time, the scale-up ratio is consistent in the scale-up experiment, which verifies the transferability of the process parameters and the implementability of industrial production.

[0127] The experimental data show that, with the increase of the substrate feed amount, the reaction conversion rate shows an upward trend; although the higher the ADP substrate feed amount, the higher the conversion rate, but considering the price factor, therefore, in the industrial production scale-up experiment in the present application, the ratio of sucrose: ADP = 175:2 is finally selected, which can not only ensure a certain conversion efficiency to meet the production demand, but also can maximize the total cost of raw materials, thereby improving the overall economic benefit of industrial production.

[0128] Example 4 Sucrose synthase (D296Q / H531L / D569Q / E610N / E663N) mutation

[0129] 1. Preparation of sucrose synthase parent plasmid

[0130] The sucrose synthase NeSUS (Genbank No. CAD85125.1) derived from Nitrosomonas europaea was codon-optimized to synthesize the required gene fragment, which was connected to the pET28a vector with Nde I and Xho I as the two end enzyme digestion sites to obtain the sucrose synthase parent pET-28a(+)-NeSUS plasmid. The obtained parent plasmid was transformed into E. coli DH5α, and after Kana screening, the colonies were picked for sequencing to determine the nucleotide sequence of the cloned sucrose synthase parent as shown in SEQ ID NO: 1, and the amino acid sequence as shown in SEQ ID NO: 2.

[0131] 2. Site-directed mutation of sucrose synthase

[0132] (1) The wild-type sucrose synthase (amino acid sequence as shown in SEQ ID NO. 1) was mutated by the method of whole plasmid site-directed mutation PCR. The sucrose synthase parent pET-28a(+)-NeSUS plasmid was used as the template, and D296Q_F / D296Q_R was used as the primer pair for the first round of site-directed mutation PCR. The primers were designed as follows:

[0133] D296Q_F: 5'-CCAAGTTGTATATATCCTGcaaCAAGTACGTGCACTGG-3'

[0134] D296Q_R: 5'-CCAGTGCACGTACTTGttgCAGGATATATACAACTTGG-3'

[0135] After PCR, 1 μL of Dpn I enzyme was added and mixed thoroughly, then incubated at 37°C for 1.0 h to digest the template plasmid. Then 5 μL of PCR reaction solution was added to the DH5α competent cells, which were placed on ice for 30 min, then heated in a 42°C water bath for 60 s, immediately placed on ice for 3 min, and then 500 μL of LB medium without antibiotics was added in the clean bench and cultured (37°C, 220 rpm, 60 min). The bacterial solution was centrifuged (4000 rpm, 5 min), and the supernatant 400 μL was discarded. The remaining bacterial solution was resuspended by blowing and sucking with a gun head, and 100 μL of bacterial solution was removed with a pipette gun and added to LB solid medium containing Kana resistance, which was evenly coated with a sterile swab. After the bacterial solution was absorbed, the plate was inverted and incubated at 37°C overnight. Each plate was inoculated with a single colony into 5.0 mL of LB medium containing 50 μg / mL Kana and cultured (37°C, 220 rpm, 6.0 h), and then sent for sequencing. After sequencing, the correct mutant plasmid pET-28a(+)-NeSUS(D296Q) was returned by the service company.

[0136] (2) The second round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(D296Q) as the template plasmid and H531L_F / H531L_R as the primer pair, and the primers were designed as follows:

[0137] H531L_F: 5'-CCGACCCGAACCGTCGTCTGctaTCTCTGATCCCGGAAATCG-3'

[0138] H531L_R: 5'-CGATTTCCGGGATCAGAGAtagCAGACGACGGTTCGGGTCGG-3'

[0139] The remaining steps were as in (1), and the mutant plasmid pET-28a(+)-NeSUS(D296Q / H531L) was obtained.

[0140] (3) The third round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(D296Q / H531L) as the template plasmid and D569Q_F / D569Q_R as the primer pair, and the primers were designed as follows:

[0141] D569Q_F: 5'-GATTTTTACCATGGCACGTCTGcaaCGCATTAAGAACATCACCG-3'

[0142] D569Q_R: 5'-CGGTGATGTTCTTAATGCGttgCAGACGTGCCATGGTAAAAATC-3'

[0143] The remaining steps were as in (1), and the mutant plasmid pET-28a(+)-NeSUS(D296Q / H531L / D569Q) was obtained.

[0144] (4) The fourth round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(D296Q / H531L / D569Q) as the template plasmid and E610N_F / E610N_R as the primer pair, and the primers were designed as follows:

[0145] E610N_F: 5'-CATTCCTCTGACCACGAAaatCAGGAACAAATCCACCG-3'

[0146] E610N_R: 5'-CGGTGGATTTGTTCCTGattTTCGTGGTCAGAGGAATG-3'

[0147] The remaining steps were as in (1), and the mutant plasmid pET-28a(+)-NeSUS(D296Q / H531L / D569Q / E610N) was obtained.

[0148] (5) The fifth round of site-directed mutagenesis was performed using pET-28a(+)-NeSUS(D296Q / H531L / D569Q / E610N) as the template plasmid and E663N_F / E663N_R as the primer pair, and the primers were designed as follows:

[0149] E663N_F: 5'-CGTGCAGCCGGCGCTGTTTaatGCATTTGGTCTGACCATC-3'

[0150] E663N_R: 5'-GATGGTCAGACCAAATGCattAAACAGCGCCGGCTGCACG-3'

[0151] The remaining steps were as in (1), and the mutant plasmid pET-28a(+)-NeSUS(D296Q / H531L / D569Q / E610N / E663N) was obtained. The nucleotide sequence of the mutant sucrose synthase (D296Q / H531L / D569Q / E610N / E663N) is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

[0152] 3. Preparation of sucrose synthase (D296Q / H531L / D569Q / E610N / E663N) mutant enzyme solution

[0153] The mutant plasmid pET-28a(+)-NeSUS(D296Q / H531L / D569Q / E610N / E663N) was transformed into E. coli BL21(DE3) to obtain the recombinant engineering bacteria E. coli BL21(DE3)-NeSUS(D296Q / H531L / D569Q / E610N / E663N). The recombinant engineering bacteria E. coli BL21(DE3)-NeSUS(D296Q / H531L / D569Q / E610N / E663N) was inoculated into 4.0 mL of LB liquid medium containing Kana resistance, and cultured at 37°C with shaking (200 rpm) overnight. The overnight culture was inoculated into 50 mL of LB liquid medium containing Kana resistance 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 mM IPTG was added to the medium, and the medium was cultured at 20-37°C with shaking for 12.0-16.0 h. After the induction was completed, 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 treatment in an ice bath, and the broken solution was centrifuged. The supernatant was collected to obtain the crude enzyme solution containing the sucrose synthase NeSUS(D296Q / H531L / D569Q / E610N / E663N) mutant.

[0154] 4. Enzyme activity determination of sucrose synthase NeSUS(D296Q / H531L / D569Q / E610N / E663N) mutant

[0155] Using sucrose 26 mg and ADP 21 mg as substrates, 0.5 mg of the crude enzyme solution of sucrose synthase NeSUS(D296Q / H531L / D569Q / E610N / E663N) mutant prepared in the third part was added, the total volume was 1 mL, the pH was adjusted to 5.0, and the reaction was carried out at 75°C with shaking at 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated.

[0156] 5. Optimization of conversion rate of sucrose synthase NeSUS(D296Q / H531L / D569Q / E610N / E663N) mutant at different reaction pH in preparation RD

[0157] Reactions were carried out at 75°C, 200 rpm, with 2.0 g of rebaudioside A as the substrate, 0.7 g of sucrose, 44.0 mg of ADP, 5.0 mg of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No. XP_015629141.1), and 5.0 mg of the crude enzyme solution of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant prepared in the third part, with a total volume of 10.0 ml. After the reaction was completed, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid sample for HPLC loading detection to calculate the conversion rate.

[0158] The conversion rates at different pH values were determined under the condition of a temperature of 75°C and a pH gradient of 4.0, 5.0, 6.0, 7.0, and 8.0, and the results are shown in the following table:

[0159] 6, Conversion rate optimization of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant at different reaction temperatures under the optimal pH (pH 6.0) in RD

[0160] Reactions were carried out at 200 rpm, with 2.0 g of rebaudioside A as the substrate, 0.7 g of sucrose, 44.0 mg of ADP, 5.0 mg of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No. XP_015629141.1), and 3.0 mg of the crude enzyme solution of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant prepared in the third part, with a total volume of 10.0 ml. After the reaction was completed, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid sample for HPLC loading detection to calculate the conversion rate.

[0161] The conversion rates at different temperatures were determined under the condition of a pH of 7.0 and a temperature gradient of 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C, and the results are shown in the following table:

[0162] 7, ADP substrate feeding amount optimization of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant

[0163] ADP 22mg, ADP 11mg, ADP 7mg, ADP 6mg, sucrose 0.7g, RA 2g, stirring to completely dissolved, adding 5.0mg of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No: XP_015629141.1) crude enzyme solution and 5.0mg sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant, each total volume of 10.0ml, adjust pH to 7. Reaction was carried out at 65℃, 200rpm for 6h respectively, after the reaction, the reaction solution was diluted 100 times with methanol, and filtered through a 0.22μm microfiltration membrane as liquid sample for HPLC sample detection.

[0164] When ADP 22mg, the conversion rate of rebaudioside D was 76.2%; when ADP 11mg, the conversion rate of rebaudioside D was 75.7%; when ADP 7mg, the conversion rate of rebaudioside D was 74.2%; when ADP 6mg, the conversion rate of rebaudioside D was 64.2%.

[0165] 8. Optimization of the amount of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant

[0166] Rebaudioside A 2.0g, sucrose 0.7g, ADP 7.0mg, stirring to completely dissolved, adding 5.0mg of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank Accession No: XP_015629141.1) crude enzyme solution and sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant in an amount of 3.0mg, 4.0mg, 5.0mg NeSUS sucrose synthase respectively, each total volume of 10.0ml, adjust pH to 7. Reaction was carried out at 65℃, 200rpm for 6h respectively, after the reaction, the reaction solution was diluted 100 times with methanol, and filtered through a 0.22μm microfiltration membrane as liquid sample for HPLC sample detection.

[0167] The conversion rate of rebaudioside D was 68.8% when the amount of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant was 3.0 mg; the conversion rate of rebaudioside D was 75.9% when the amount of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant was 4.0 mg; and the conversion rate of rebaudioside D was 75.2% when the amount of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant was 5.0 mg.

[0168] 9. pH stability analysis of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant

[0169] pH stability: The sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant enzyme solution was placed in a buffer with a pH of 5.5-9.5, and after 24 hours of incubation at 4°C, the residual enzyme activity was measured. The results showed that the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant maintained more than 70% enzyme activity in the pH range of 5-7, while the wild type only maintained the same level in the pH range of 5.5-6.5.

[0170] The wild type is only stable in the pH range of 5.5-6.5, while the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant has a wider stable range and is more suitable for the neutral reaction system in some industrial scenarios (such as the natural pH of some buffers close to 7.0).

[0171] Example 1: Enzyme activity test of sucrose synthase parent

[0172] 1. Preparation of sucrose synthase parent plasmid

[0173] The sucrose synthase NeSUS (Genbank No. CAD85125.1) derived from Nitrosomonas europaea was codon-optimized to synthesize the required gene fragment, which was connected to the pET28a vector with Nde I and Xho I as the two end enzyme digestion sites, to obtain the sucrose synthase parent pET-28a(+)-NeSUS plasmid. The obtained parent plasmid was then transformed into E. coli DH5α, and after Kana screening, colonies were picked for sequencing to determine the nucleotide sequence of the cloned sucrose synthase parent, which is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0174] 2. Preparation of sucrose synthase parent enzyme solution

[0175] The parent plasmid was transformed into E. coli BL21 (DE3) to obtain recombinant engineering bacteria E. coli BL21 (DE3)-NeSUS. The recombinant engineering bacteria E. coli BL21 (DE3)-NeSUS was inoculated into 4.0 mL liquid LB medium at a proportion of 1%, and was cultured at 37°C with shaking (200 rpm) overnight. The overnight culture was inoculated into a large volume of liquid LB medium at a proportion of 1%, and was cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. Then, 0.1 mM-1 mM IPTG was added, and the culture was incubated at 20-37°C with shaking for 12.0-16.0 h. After the induction, the bacteria were collected by centrifugation, and the cells were resuspended in 50 mM phosphate buffer (pH 7.2). The cells were broken by ultrasonic treatment in an ice bath, and the broken solution was centrifuged. The supernatant was collected to obtain a crude enzyme solution containing sucrose synthase parent mutants.

[0176] 3. Enzyme activity determination of sucrose synthase parent

[0177] Using sucrose 26 mg and ADP 21 mg as substrates, 0.5 mg of the crude enzyme solution of sucrose synthase NeSUS prepared in the second part was added, the total volume was 1 mL, the pH was adjusted to 5, and the reaction was carried out at 75°C with shaking at 200 rpm for 30 min. Then, the sample was taken for HPLC detection, and the enzyme activity was calculated.

[0178] 4. Reaction temperature determination of sucrose synthase parent

[0179] 5. Enzyme activity test results of sucrose synthase mutants and their parents are as follows:

[0180] As shown in the above table, the enzyme activity of the mutant of the present application is increased by 12%-38% compared with the wild type, and the rebaudioside D conversion rate is as high as 84.2% (in Example 3, under the conditions of 60°C, pH 6.0, and 6 h), and the stability is good in the pH range of 5.0-7.0 and the temperature range of 55-75°C.

[0181] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, some modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims. SEQ ID NO. 1 Nitrosomonas europaea sucrose synthase nucleotide sequence

[0182] SEQ ID NO. 2 Nitrosomonas europaea sucrose synthase amino acid sequence

[0183] SEQ ID NO. 3 Nitrosomonas europaea sucrose synthase (R567T / R636Q / A642N) mutant nucleotide sequence

[0184] SEQ ID NO. 4 Nitrosomonas europaea sucrose synthase (R567T / R636Q / A642N) mutant amino acid sequence

[0185] SEQ ID NO. 5 Nitrosomonas europaea sucrose synthase (G514L / R567T / A642N / T742R) mutant nucleotide sequence

[0186] SEQ ID NO. 6 Nitrosomonas europaea sucrose synthase (G514L / R567T / A642N / T742R) mutant amino acid sequence

[0187] SEQ ID NO. 7 Nitrosomonas europaea sucrose synthase (G514L / R567T / A642N / L667T / T742R) mutant nucleotide sequence

[0188] SEQ ID NO. 8 Nitrosomonas europaea sucrose synthase (G514L / R567T / A642N / L667T / T742R) mutant amino acid sequence

[0189] SEQ ID NO. 9 Nitrosomonas europaea sucrose synthase (D296Q / H531L / D569Q / E610N / E663N) mutant nucleotide sequence

[0190] SEQ ID NO. 10 Nitrosomonas europaea sucrose synthase (D296Q / H531L / D569Q / E610N / E663N) mutant amino acid sequence

Claims

A sucrose synthase mutant characterized in that, The sucrose synthase mutant is capable of regenerating ADP-glucose from sucrose and ADP to catalyze the conversion of rebaudioside A to rebaudioside D in cooperation with the β-1,2-glucosidase; the sucrose synthase mutant contains mutations in at least one of the following sites based on the amino acid sequence shown in SEQ ID NO: 2: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R. The sucrose synthase mutant according to claim 1, characterized in that The amino acid sequence of the sucrose synthase mutant comprises one of the following combinations of mutations based on the wild-type sucrose synthase shown in SEQ ID NO: 2: R567T, R636Q and A642N; G514L, R567T, A642N and T742R; G514L, R567T, A642N, L667T and T742R; D296Q, H531L, D569Q, E610N and E663N. The sucrose synthase mutant according to claim 2, characterized in that The nucleotide sequence of the sucrose synthase mutant is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:

6. The sucrose synthase mutant according to claim 3, characterized in that The β-1,2-glucosidase is a UDP-glucosyltransferase mutant. A method for preparing rebaudioside D using a sucrose synthase mutant, characterized in that, The sucrose synthase mutant and the β-1,2-glucosidase of claims 1-4 are used as catalysts to generate rebaudioside D from rebaudioside A, sucrose and ADP. The method for preparing rebaudioside D using the sucrose synthase mutant according to claim 5, wherein, The reaction temperature in the catalytic reaction is controlled at 55-80°C, the pH is 4.0-8.0, and the reaction time is 0.5-18h. The method for preparing rebaudioside D according to claim 5 or the sucrose synthase mutant, characterized in that, The reaction temperature in the catalytic reaction is controlled at 55-75°C, the pH is 5.0-7.0, and the reaction time is 4-18h. The method for preparing rebaudioside D using the sucrose synthase mutant according to claim 5, wherein, The reaction temperature in the catalytic reaction is controlled at 65-70°C, the pH is 6.0, and the reaction time is 6h. The method for preparing rebaudioside D using the sucrose synthase mutant according to any one of claims 5 to 8, characterized in that, The reaction is carried out in a solution comprising the following components: The initial reaction concentration of rebaudioside A is 160-300g / L, The initial reaction concentration of sucrose is 60-80g / L, The initial reaction concentration of ADP is 0.4-4.4g / L, The initial reaction concentration of β-1,2-glucosidase is 0.4-0.6g / L, The total protein concentration of the crude enzyme solution of the NeSUS sucrose synthase mutant of any one of claims 1-4 in the reaction system is 0.3-0.5g / L, and the balance is solvent. The method for preparing rebaudioside D using the sucrose synthase mutant according to any one of claims 5 to 7, wherein, The reaction is carried out in a solution comprising the following components: The initial reaction concentration of rebaudioside A is 200g / L, The initial reaction concentration of sucrose is 70g / L, The initial reaction concentration of ADP is 0.7g / L, The initial reaction concentration of β-1,2-glucosidase is 0.5g / L, The total protein concentration of the crude enzyme solution of the NeSUS sucrose synthase mutant of any one of claims 1-4 in the reaction system is 0.5g / L, and the balance is pure water; The pH is adjusted to 6.0, the reaction is carried out at 60°C for 18h, and rebaudioside D is generated by catalytic reaction.

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