Sucrose synthase mutant and method for preparing rd using the same

Abstract

The application discloses a sucrose synthetase mutant and a method for preparing RD by using the sucrose synthetase mutant, wherein the sucrose synthetase mutant contains at least one of the following mutations: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R on the basis of the amino acid sequence shown in SEQ ID NO:2; the sucrose synthetase mutant has higher enzyme activity than the wild type, and meanwhile, some mutants still maintain a high conversion rate in a wider pH range.

Description

Technical Field

[0001] This invention relates to the field of steviol glycoside production technology, and in particular to a sucrose synthase mutant and a method for preparing RD. Background Technology

[0002] Stevioside is a natural glycoside sweetener extracted from the leaves of the stevia plant (Stevioside is a member of the Asteraceae family). Its chemical formula is C6H2O. 38 H 60 O 18 With CAS number 57817-89-7, it is characterized by low calories (only 1 / 300 the caloric value of sucrose), high sweetness (about 300 times that of sucrose), and strong stability. Data from the international sweetener industry shows that steviol glycosides are widely used in the production of food, beverages, and seasonings in Asia, North America, South America, and the European Union.

[0003] Reb D (also known as Rebaudioside D) is a high-sweetness, low-calorie natural sweetener. It stands out among many natural sweeteners due to its higher sweetness, less bitter aftertaste, and sucrose-like texture. Initially, RD was mainly extracted from the plant stevia, but its proportion in stevia was less than 1%, making this method costly and unable to meet market demand. Currently, the main synthetic methods for RD include chemical methods, fermentation methods, and bio-enzymatic methods. Biocatalytic methods yield high concentrations of RD while reducing environmental pollution and increasing the yield of the desired RD product.

[0004] Sucrose synthase (SuSy) is an enzyme found in plants and some microorganisms that plays a crucial role in sucrose metabolism. It primarily catalyzes the synthesis and breakdown of sucrose, specifically the conversion of ADP to ADPG. This process is not only a core step in carbohydrate metabolism but also indirectly provides the material basis for the conversion of Reb A (Reb A) to Reb D (Reb D) by offering a key glycosyl donor. The activity of SuSy directly determines the concentration level of ADPG in the system, while the availability of ADPG becomes a key factor limiting the efficiency of Reb A glycosylation.

[0005] Sucrose synthases catalyze the conversion of ADP to ADPG, thereby accelerating the rate and efficiency of rebaudioside A to rebaudioside D. However, wild-type sucrose synthases exhibit low catalytic activity, limited affinity for ADP and ADPG generation efficiency, and their catalytic direction is more inclined towards sucrose synthesis, leading to insufficient ADPG supply and limiting the efficient synthesis of Reb D. Therefore, genetic modification techniques are needed to mutate wild-type sucrose synthases to further enhance their activity, providing sufficient substrate for the glycosylation reaction of Reb A to Reb D, and thus improving the synthesis efficiency of Reb D. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention utilizes a mutant of sucrose synthase to further enhance its activity, thereby solving the problem of low catalytic efficiency of wild-type sucrose synthase, improving the conversion efficiency of RA to RD, and achieving efficient, energy-saving, and large-scale production of rebaudioside D.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

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

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

[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, and SEQ ID NO:6.

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

[0016] A method for preparing rebaudioside D using a sucrose synthase mutant involves using a sucrose synthase mutant and β-1,2-glucosidase as catalysts, and rebaudioside A, sucrose, and ADP 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 0.5h-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 4h-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 160-300 g / L, the initial reaction concentration of sucrose is 60-80 g / L, the initial reaction concentration of ADP is 0.4-4.4 g / L, the initial reaction concentration of β-1,2-glucosidase is 0.4-0.6 g / L, the total protein concentration of the crude enzyme solution of the NeSUS sucrose synthase mutant in the reaction system is 0.3-0.5 g / L, and the remainder 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 200 g / L, the initial reaction concentration of sucrose is 70 g / L, the initial reaction concentration of ADP is 0.7 g / L, the initial reaction concentration of β-1,2-glucosidase is 0.5 g / L, the total protein concentration of the crude enzyme solution of the NeSUS sucrose synthase mutant in the reaction system is 0.5 g / L, and the remainder is pure water; the pH is adjusted to 6.0, and the reaction is carried out at 60°C for 18 h to catalyze the reaction to generate rebaudioside D. Beneficial effects

[0022] The sucrose synthase mutant in this application can utilize sucrose and ADP to regenerate ADP-glucose, thereby synergistically catalyzing the conversion of rebaudioside A to rebaudioside D by β-1,2-glucosidase.

[0023] Specifically, the sucrose synthase mutant of the present invention has an enzyme activity that is 12% to 38% higher than that of the wild type (see the enzyme activity test results of the sucrose synthase mutant and its parent in the comparative example), significantly improves the conversion efficiency, and has a stronger adaptability to reaction conditions. Some mutants maintain a high conversion rate over a wide pH range. This characteristic of the mutant enhances its applicability and feasibility in industrial production and reduces the impact of fluctuations in reaction conditions on the reaction results. Attached Figure Description

[0024] Figure 1 The HPLC chromatogram of the synthesis of rebaudioside D catalyzed by the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) of Example 3 of the present invention (reaction time 0 hours).

[0025] Figure 2 HPLC chromatogram of the synthesis of rebaudioside D catalyzed by the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) in Example 3 of the present invention (reaction time 1 hour).

[0026] Figure 3 HPLC chromatogram of the synthesis of rebaudioside D catalyzed by the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) in Example 3 of the present invention (reaction time 2 hours).

[0027] Figure 4 HPLC chromatogram of the synthesis of rebaudioside D catalyzed by the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) in Example 3 of the present invention (reaction time 18 hours). Detailed Implementation

[0028] To facilitate understanding of the present invention, it will be described in detail below with reference to specific embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[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 invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice of this invention, preferred methods and materials are now described.

[0030] TB medium: yeast extract 23.6 g / L, tryptone 11.8 g / L, dipotassium hydrogen phosphate 9.4 g / L, potassium dihydrogen phosphate 2.2 g / L, glycerol 4.0 mL / L.

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

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

[0033] PCR setup: initial denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 12 min, 18 cycles, final extension at 68°C for 10 min, and hold at 12°C.

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

[0035] 1. Preparation of sucrose synthase parent plasmids

[0036] derived from Nitrosaminoglycans Nitrosomonas europaea Sucrose synthase Ne After codon optimization of SUS (Genbank accession number CAD85125.1), the required gene fragment was synthesized and ligated into the pET28a vector, with Nde I and Xho I restriction enzyme sites at both ends, to obtain the sucrose synthase parent pET-28a(+)- NeSUS The plasmid was then transformed into E. coli DH5α. After Kana screening, colonies were selected and sequenced to determine the nucleotide sequence of the cloned sucrose synthase parent, as shown in SEQ ID NO: 1, and its amino acid sequence, as shown in SEQ ID NO: 2.

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

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

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

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

[0041] After PCR, 1 μL of Dpn I enzyme was added and thoroughly mixed. The mixture was then incubated at 37 °C for 1.0 h to digest the template plasmid. Subsequently, 5 μL of PCR reaction solution was added to DH5α competent cells, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42 °C water bath for 60 s, immediately returned to ice for 3 min, and incubated in a clean bench with 500 μL of antibiotic-free LB medium (37 °C, 220 rpm, 60 min). The culture was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining culture was resuspended using a pipette tip. 100 μL of the culture was transferred to LB solid medium containing Kana resistance in a clean bench and gently spread with a sterile spreader. After absorption, the plates were inverted and incubated overnight at 37 °C. A single colony from each plate was inoculated into 5.0 mL of medium containing 50 μg / mL Kana. After culturing in LB medium (37℃, 220 rpm, 6.0 h), the samples were sent for sequencing. Upon completion of sequencing, the service company returned the correct mutant plasmid pET-28a(+)-. Ne SUS (R567T).

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

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

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

[0045] The remaining steps are as follows (1), to obtain the mutant plasmid pET-28a(+)-NeSUS(R567T / R636Q).

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

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

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

[0049] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)-NeSUS (R567T / R636Q / A642N), the nucleotide sequence of the mutant sucrose synthase (R567T / R636Q / A642N) 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) was transformed into E. coli BL21(DE3) to obtain the recombinant engineered strain E. coli BL21(DE3)-NeSUS(R567T / R636Q / A642N). A single colony of the recombinant engineered strain E. coli BL21(DE3)-NeSUS(R567T / R636Q / A642N) was inoculated into 4.0 mL of Kana-resistant LB broth and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculum to 50 mL of Kana-resistant LB broth and cultured at 37°C with shaking (200 rpm) until OD500. 600 When the pH reaches 0.6-0.8, add 0.1 mM to 1 mM IPTG to a final concentration and incubate at 20-37℃ with shaking for 12.0-16.0 h. After induction, collect the cells by centrifugation, resuspend the cells in 50 mM phosphate buffer (pH 7.2), and sonicate the cells in an ice bath. Centrifuge the lysate and collect the supernatant to obtain the crude enzyme solution containing the sucrose synthase NeSUS (R567T / R636Q / A642N) mutant.

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

[0053] Using 26 mg sucrose and 21 mg ADP as substrates, 0.5 mg of crude enzyme solution of the NeSUS (R567T / R636Q / A642N) mutant sucrose synthase prepared in Part 3 was added, bringing the total volume to 1 ml. The pH was adjusted to 5.0, and the reaction was carried out at 75 °C and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity.

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

[0055] Using 2.0 g of rebaudioside A as a substrate, 0.7 g of sucrose, 44.0 mg of ADP, and 5.0 mg of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) prepared in Part 3, along with 5.0 mg of crude enzyme solution of sucrose synthase NeSUS (R567T / R636Q / A642N) mutant prepared in Part 3, the total volume was 10.0 ml. The reaction was carried out at 75℃ and 200 rpm. After the reaction, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid phase sample for HPLC analysis to calculate the conversion rate.

[0056] At a temperature of 75℃, pH gradients of 4.0, 5.0, 6.0, 7.0, and 8.0 were set, and the conversion rates at different pH values ​​were measured. The results are shown in the table below:

[0057]

[0058] Example 2: Sucrose synthase (G514L / R567T / A642N / T742R) mutation

[0059] 1. Preparation of sucrose synthase parent plasmids

[0060] derived from Nitrosaminoglycans Nitrosomonas europaea Sucrose synthase Ne After codon optimization of SUS (Genbank accession number CAD85125.1), the required gene fragment was synthesized and ligated into the pET28a vector, with Nde I and Xho I restriction enzyme sites at both ends, to obtain the sucrose synthase parent pET-28a(+)- Ne The SUS plasmid was then transformed into E. coli DH5α. After Kana screening, colonies were selected and sequenced to determine the nucleotide sequence of the cloned sucrose synthase parent as shown in SEQ ID NO: 1, and its amino acid sequence as shown in SEQ ID NO: 2.

[0061] 2. Site-directed mutagenesis of sucrose synthase

[0062] (1) The wild-type sucrose synthase (amino acid sequence as shown in SEQ ID NO. 1) was mutated using the whole plasmid site-directed mutagenesis PCR method, with the sucrose synthase parent pET-28a(+)- Ne Using SUS plasmid as a template, the first round of site-directed mutagenesis PCR was performed with G514L_F / G514L_R primer pair. The primers were designed as follows:

[0063] G514L_F:5'-CAACATCGTATCCCCGcttGCTAACGCAGACATTTATTTTC -3'

[0064] G514L_R: 5'-GAAAATAAATGTCTGCGTTAGCaagCGGGGATACGATGTTG-3'

[0065] After PCR, 1 μL of Dpn I enzyme was added and thoroughly mixed. The mixture was then incubated at 37 ℃ for 1.0 h to digest the template plasmid. Subsequently, 5 μL of PCR reaction solution was added to DH5α competent cells, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42 ℃ water bath for 60 s, immediately returned to ice for 3 min, and cultured in a clean bench with 500 μL of antibiotic-free LB medium (37 ℃, 220 rpm, 60 min). The culture was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining culture was resuspended using a pipette tip. 100 μL of the culture was transferred to LB solid medium containing Kana resistance in a clean bench and gently spread with a sterile spreader. After absorption, the plates were inverted and incubated overnight at 37 ℃. A single colony from each plate was picked and inoculated into 5.0 mL of LB medium containing 50 μg / mL Kana and cultured at 37 ℃. Samples were sent for sequencing after being set at ℃, 220 rpm, and 6.0 h. After sequencing, the service company returned the correct mutant plasmid pET-28a(+)-. Ne SUS (G514L).

[0066] (2) pET-28a(+)- Ne Using SUS(G514L) as the template plasmid, a second round of site-directed mutagenesis was performed using the R567T_F / R567T_R primer pair. The primers were designed as follows:

[0067] R567T_F: 5'-GCTGATTTTTACCATGGCAactCTGGATCGCATTAAGAAC -3'

[0068] R567T_R:5'-GTTCTTAATGCGATCCAGagtTGCCATGGTAAAAATCAGC -3'

[0069] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne SUS (G514L / R567T).

[0070] (3) pET-28a(+)- Ne Using SUS(G514L / R567T) as the template plasmid, and A642N_F / A642N_R as the primer pair, a third round of site-directed mutagenesis was performed. The primers were designed as follows:

[0071] A642N_F:5'-CTGGACAAAAACCTGaatGGCGAACTGTATCGC-3'

[0072] A642N_R:5'-GCGATACAGTTCGCCattCAGGTTTTTGTCCAG-3'

[0073] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne SUS (G514L / R567T / A642N)

[0074] (4) pET-28a(+)- Ne Using SUS(G514L / R567T / A642N) as the template plasmid, and T742R_F / T742R_R as the primer pair, a fourth round of site-directed mutagenesis was performed. The primers were designed as follows:

[0075] T742R_F: 5'-GTGTAGCGTCTCGTTACagaTGGAAGCTGTATGCCGAAC-3'

[0076] T742R_R:5'-GTTCGGCATACAGCTTCCAtctGTAACGAGACGCTACAC-3'

[0077] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne The nucleotide sequence of the mutant SUS (G514L / R567T / A642N / T742R) sucrose synthase (G514L / R567T / A642N / T742R) is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0078] 3. Preparation of sucrose synthase (G514L / R567T / A642N / T742R) mutant enzyme solution

[0079] The mutant plasmid pET-28a(+)-NeSUS(G514L / R567T / A642N / T742R) was transformed into E. coli BL21(DE3) to obtain the recombinant engineered strain E. coli BL21(DE3)-NeSUS(G514L / R567T / A642N / T742R). A single colony of the recombinant engineered E. coli BL21(DE3)-NeSUS (G514L / R567T / A642N / T742R) was inoculated into 4.0 mL of LB liquid medium containing Kana resistance and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculum to 50 mL of LB liquid medium containing Kana resistance and cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. Finally, 0.1 mM to 1 mM IPTG was added and cultured at 20-37°C with shaking for 12.0-16.0 h. After induction, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.2). The cells were then sonicated in an ice bath to disrupt them. The disrupted solution was centrifuged and the supernatant was collected to obtain the crude enzyme solution containing the sucrose synthase NeSUS (G514L / R567T / A642N / T742R) mutant.

[0080] 4. Enzyme activity assay of the sucrose synthase NeSUS (G514L / R567T / A642N / T742R) mutant

[0081] Using 26 mg sucrose and 21 mg ADP as substrates, 0.5 mg of crude enzyme solution of the sucrose synthase NeSUS (G514L / R567T / A642N / T742R) mutant prepared in Part 3 was added, bringing the total volume to 1 ml. The pH was adjusted to 5.0, and the reaction was carried out at 75℃ and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity.

[0082] 5. Preparation of sucrose synthase NeSUS (G514L / R567T / A642N / T742R) mutant and optimization of conversion rate at different reaction pH in RD.

[0083] Using rebaudioside A 2.0 g as substrate, sucrose 0.7 g, ADP 44.0 mg, and UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) 5.0 mg, and then adding crude enzyme solution of the sucrose synthase NeSUS (G514L / R567T / A642N / T742R) mutant prepared in Part 3, the total volume was 10.0 ml. The reaction was carried out at 75℃ and 200 rpm. After the reaction, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid phase sample for HPLC analysis to calculate the conversion rate.

[0084] At a temperature of 75℃, pH gradients of 4.0, 5.0, 6.0, 7.0, and 8.0 were set, and the conversion rates at different pH values ​​were measured. The results are shown in the table below:

[0085]

[0086] Example 3: Mutation of sucrose synthase (G514L / R567T / A642N / L667T / T742R)

[0087] 1. Preparation of sucrose synthase parent plasmids

[0088] derived from Nitrosaminoglycans Nitrosomonas europaea Sucrose synthase Ne After codon optimization of SUS (Genbank accession number CAD85125.1), the required gene fragment was synthesized and ligated into the pET28a vector, with Nde I and Xho I restriction enzyme sites at both ends, to obtain the sucrose synthase parent pET-28a(+)- Ne The SUS plasmid was then transformed into E. coli DH5α. After Kana screening, colonies were selected and sequenced to determine the nucleotide sequence of the cloned sucrose synthase parent as shown in SEQ ID NO: 1, and its amino acid sequence as shown in SEQ ID NO: 2.

[0089] 2. Site-directed mutagenesis of sucrose synthase

[0090] (1) The wild-type sucrose synthase (amino acid sequence as shown in SEQ ID NO. 1) was mutated using the whole plasmid site-directed mutagenesis PCR method, with the sucrose synthase parent pET-28a(+)- Ne Using SUS plasmid as a template, the first round of site-directed mutagenesis PCR was performed using G514L_F / G514L_R primer pairs. The primers were designed as follows:

[0091] G514L_F:5'-CAACATCGTATCCCCGcttGCTAACGCAGACATTTATTTTC -3'

[0092] G514L_R: 5'-GAAAATAAATGTCTGCGTTAGCaagCGGGGATACGATGTTG-3'

[0093] After PCR, 1 μL of Dpn I enzyme was added and thoroughly mixed. The mixture was then incubated at 37 °C for 1.0 h to digest the template plasmid. Subsequently, 5 μL of PCR reaction solution was added to DH5α competent cells, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42 °C water bath for 60 s, immediately returned to ice for 3 min, and cultured in a clean bench with 500 μL of antibiotic-free LB medium (37 °C, 220 rpm, 60 min). The culture was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining culture was resuspended using a pipette tip. 100 μL of the culture was transferred to LB solid medium containing Kana resistance in a clean bench and gently spread with a sterile spreader. After absorption, the plates were inverted and incubated overnight at 37 °C. A single colony from each plate was picked and inoculated into 5.0 mL of LB medium containing 50 μg / mL Kana and cultured at 37 °C. Samples were sent for sequencing after being set at ℃, 220 rpm, and 6.0 h. After sequencing, the service company returned the correct mutant plasmid pET-28a(+)-. Ne SUS (G514L).

[0094] (2) pET-28a(+)- Ne Using SUS(G514L) as the template plasmid, a second round of site-directed mutagenesis was performed using the R567T_F / R567T_R primer pair. The primers were designed as follows:

[0095] R567T_F:5'-GCTGATTTTTACCATGGCAactCTGGATCGCATTAAGAAC-3'

[0096] R567T_R:5'-GTTCTTAATGCGATCCAGagtTGCCATGGTAAAAATCAGC-3'

[0097] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne SUS (G514L / R567T).

[0098] (3) pET-28a(+)- NeUsing SUS(G514L / R567T) as the template plasmid, and A642N_F / A642N_R as the primer pair, a third round of site-directed mutagenesis was performed. The primers were designed as follows:

[0099] A642N_F:5'-CTGGACAAAAACCTGaatGGCGAACTGTATCGC-3'

[0100] A642N_R:5'-GCGATACAGTTCGCCattCAGGTTTTTGTCCAG-3'

[0101] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne SUS (G514L / R567T / A642N).

[0102] (4) pET-28a(+)- Ne Using SUS (G514L / R567T / A642N) as the template plasmid, and L667T_F / L667T_R as the primer pair, a fourth round of site-directed mutagenesis was performed. The primers were designed as follows:

[0103] L667T_F:5'-GTTTGAAGCATTTGGTactACCATCATTGAGGCTATGG-3'

[0104] L667T_R:5'-CCATAGCCTCAATGATGTGTagtACCAAATGCTTCAAAC-3'

[0105] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne SUS (G514L / R567T / A642N / L667T).

[0106] (5) pET-28a(+)- Ne Using SUS(G514L / R567T / A642N / L667T) as the template plasmid, and T742R_F / T742R_R as the primer pair, a fifth round of site-directed mutagenesis was performed. The primers were designed as follows:

[0107] T742R_F: 5'-GTGTAGCGTCTCGTTACagaTGGAAGCTTGTATGCCGAAC -3'

[0108] T742R_R: 5'-GTGTAGCGTCTCGTTACagaTGGAAGCTTGTATGCCGAAC-3'

[0109] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)-Ne The nucleotide sequence of the mutant SUS (G514L / R567T / A642N / L667T / T742R) sucrose synthase (G514L / R567T / A642N / L667T / T742R) is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8.

[0110] 3. Preparation of sucrose synthase (G514L / R567T / A642N / L667T / T742R) mutant enzyme solution

[0111] The mutant plasmid pET-28a(+)- Ne Recombinant engineered E. coli BL21(DE3) was obtained by transforming SUS (G514L / R567T / A642N / L667T / T742R) into E. coli BL21(DE3). Ne SUS (G514L / R567T / A642N / L667T / T742R). Recombinant engineered E. coli BL21(DE3)- Ne Single colonies of SUS (G514L / R567T / A642N / L667T / T742R) were inoculated into 4.0 mL of LB broth containing Kana resistance and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculum to 50 mL of LB broth containing Kana resistance and cultured at 37°C with shaking (200 rpm) until the OD600 reached 0.6-0.8. Finally, 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 induction, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.2). The cells were then sonicated in an ice bath, and the lysate was centrifuged. The supernatant was collected to obtain the sucrose synthase. Ne Crude enzyme solution of SUS (G514L / R567T / A642N / L667T / T742R) mutant.

[0112] 4. Sucrose synthase Ne Enzyme activity assay of SUS (G514L / R567T / A642N / L667T / T742R) mutant

[0113] Using 26 mg sucrose and 21 mg ADP as substrates, 0.5 mg of crude enzyme solution of the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant prepared in Part 3 was added, bringing the total volume to 1 ml. The pH was adjusted to 5.0, and the reaction was carried out at 75℃ and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity.

[0114] 5. Sucrose synthase Ne Preparation of SUS(G514L / R567T / A642N / L667T / T742R) mutants; optimization of conversion rates at different reaction pH in RD.

[0115] Using rebaudioside A 2.0 g as substrate, sucrose 0.7 g, ADP 44.0 mg, and UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) 5.0 mg were added, along with 5.0 mg of crude enzyme solution of the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant prepared in Part 3, for a total volume of 10.0 ml. The reaction was carried out at 75 °C and 200 rpm. After the reaction, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid phase sample for HPLC analysis to calculate the conversion rate.

[0116] At a temperature of 75℃, pH gradients of 4.0, 5.0, 6.0, 7.0, and 8.0 were set, and the conversion rates at different pH values ​​were measured. The results are shown in the table below:

[0117]

[0118] 6. Preparation of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant and optimization of conversion rate at different reaction temperatures under optimal pH (pH 6.0) in RD.

[0119] Using rebaudioside A 2.0 g as substrate, sucrose 0.7 g, ADP 44.0 mg, and UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) 5.0 mg, along with crude enzyme solution of the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant prepared in Part 3, a total volume of 10.0 ml was added. The reaction was carried out at 200 rpm. After the reaction, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid phase sample for HPLC analysis to calculate the conversion rate.

[0120] Under pH 6.0 conditions, temperature gradients of 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃ were set, and the conversion rates at different temperatures were measured. The results are shown in the table below:

[0121]

[0122] 7. Optimization of ADP substrate feed rate for preparation of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant

[0123] Prepare separate solutions of ADP 44 mg, ADP 18 mg, ADP 8 mg, and ADP 4 mg, sucrose 0.7 g, and RA 2 g, respectively, and stir until completely dissolved. Add 5.0 mg of crude enzyme solution of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) and 5.0 mg of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant, with a total volume of 10.0 ml for each solution, and adjust the pH to 6. Incubate the solutions at 60℃ and 200 rpm for 6 h. After the reaction, dilute the reaction solution 100 times with methanol, filter through a 0.22 μm microfiltration membrane, and use as a liquid phase sample for HPLC analysis.

[0124] The conversion rate of rebaudioside D was 87.1% when ADP was 44 mg; 85.6% when ADP was 18 mg; 84.2% when ADP was 8 mg; and 77.6% when ADP was 4 mg.

[0125] 8. Optimization of dosage of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant

[0126] Prepare a solution of 2.0 g of rebaudioside A, 0.7 g of sucrose, and 8 mg of ADP, stirring until completely dissolved. Add 5.0 mg of crude enzyme solution of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) and 3.0 mg, 4.0 mg, and 5.0 mg of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant, respectively, with a total volume of 10.0 ml per solution. Adjust the pH to 6. Incubate the reaction solution at 60 °C and 200 rpm for 18 h. After the reaction, dilute the reaction solution 100 times with methanol, filter through a 0.22 μm microfiltration membrane, and use as a liquid phase sample for HPLC analysis.

[0127] When the dosage of the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant was 3.0 mg, the conversion rate of rebaudioside D was 84.6%; when the dosage of the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant was 4.0 mg, the conversion rate of rebaudioside D was 82.3%; and when the dosage of the sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant was 5.0 mg, the conversion rate of rebaudioside D was 81%.

[0128] 9. Scale-up experiment for the preparation of rebaudioside D from sucrose synthase mutant.

[0129] Rebaudioside A 6.0 kg, sucrose 2.1 kg, ADP 2.4 g, and UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) 15.0 g were added, with a total volume of 30.0 L. The mixture was stirred until completely dissolved. Then, 9 g of crude enzyme solution of sucrose synthase NeSUS (G514L / R567T / A642N / L667T / T742R) mutant was added, and the pH was adjusted to 6.0. The reaction was carried out at 60 °C. After the reaction, the reaction solution was diluted 100-fold with methanol and filtered through a 0.22 μm microfiltration membrane for HPLC analysis. After 18 h of reaction, 82.1% conversion was achieved.

[0130] From the 10mL small-scale test in the examples to the 30L scale-up experiment, the feed amount of rebaudioside A increased from 2.0g to 6.0kg, and the conversion rate decreased from 84.2% to 82.1%, a decrease of only 2.1%, indicating high process stability. Simultaneously, the consistent scale-up ratio in the scale-up experiments verified the transferability of process parameters and the feasibility of industrial-scale production.

[0131] Experimental data show that the reaction conversion rate increases with the increase of substrate feed amount. Although the conversion rate is higher when the ADP substrate feed amount is higher, the industrial-scale production scale-up experiment in this invention finally selected a ratio of sucrose:ADP = 175:2, which can ensure a certain conversion efficiency to meet production needs, and minimize the total cost of raw materials, thereby improving the overall economic benefits of industrial production.

[0132] Example 4: Mutation of sucrose synthase (D296Q / H531L / D569Q / E610N / E663N)

[0133] 1. Preparation of sucrose synthase parent plasmids

[0134] derived from Nitrosaminoglycans Nitrosomonas europaea Sucrose synthase Ne After codon optimization of SUS (Genbank accession number CAD85125.1), the required gene fragment was synthesized and ligated into the pET28a vector, with Nde I and Xho I restriction enzyme sites at both ends, to obtain the sucrose synthase parent pET-28a(+)- Ne The SUS plasmid was then transformed into E. coli DH5α. After Kana screening, colonies were selected and sequenced to determine the nucleotide sequence of the cloned sucrose synthase parent as shown in SEQ ID NO: 1, and its amino acid sequence as shown in SEQ ID NO: 2.

[0135] 2. Site-directed mutagenesis of sucrose synthase

[0136] (1) The wild-type sucrose synthase (amino acid sequence as shown in SEQ ID NO. 1) was mutated using the whole plasmid site-directed mutagenesis PCR method, with the sucrose synthase parent pET-28a(+)- Ne Using SUS plasmid as a template, and D296Q_F / D296Q_R as the primer pair, the first round of site-directed mutagenesis PCR was performed. The primers were designed as follows:

[0137] D296Q_F:5'-CCAAGTTGTATATCCTGcaaCAAGTACGTGCACTGG -3'

[0138] D296Q_R: 5'-CCAGTGCACGTACTTGttgCAGGATATATACAACTTGG-3'

[0139] After PCR, 1 μL of Dpn I enzyme was added and thoroughly mixed. The mixture was then incubated at 37 °C for 1.0 h to digest the template plasmid. Subsequently, 5 μL of PCR reaction solution was added to DH5α competent cells, and the cells were incubated on ice for 30 min. The cells were then heat-shocked in a 42 °C water bath for 60 s, immediately returned to ice for 3 min, and cultured in a clean bench with 500 μL of antibiotic-free LB medium (37 °C, 220 rpm, 60 min). The culture was centrifuged (4000 rpm, 5 min), and 400 μL of supernatant was discarded. The remaining culture was resuspended using a pipette tip. 100 μL of the culture was transferred to LB solid medium containing Kana resistance in a clean bench and gently spread with a sterile spreader. After absorption, the plates were inverted and incubated overnight at 37 °C. A single colony from each plate was picked and inoculated into 5.0 mL of LB medium containing 50 μg / mL Kana and cultured at 37 °C. Samples were sent for sequencing after being set at ℃, 220 rpm, and 6.0 h. After sequencing, the service company returned the correct mutant plasmid pET-28a(+)-.Ne SUS (D296Q).

[0140] (2) pET-28a(+)- Ne Using SUS(D296Q) as the template plasmid, and H531L_F / H531L_R as the primer pair, a second round of site-directed mutagenesis was performed. The primers were designed as follows:

[0141] H531L_F:5'-CCGACCCGAACCGTCGTCTGctaTCTCTGATCCCGGAAATCG -3'

[0142] H531L _R: 5'-CGATTTCCGGGATCAGAGAtagCAGACGACGGTTCGGGTCGG -3'

[0143] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne SUS (D296Q / H531L).

[0144] (3) pET-28a(+)- Ne Using SUS(D296Q / H531L) as the template plasmid, and D569Q_F / D569Q_R as the primer pair, a third round of site-directed mutagenesis was performed. The primers were designed as follows:

[0145] D569Q_F:5'-GATTTTTACCATGGCACGTCTGcaaCGCATTAAGAACATCACCG-3'

[0146] D569Q_R:5'-CGGTGATGTTCTTAATGCGttgCAGACGTGCCATGGTAAAAATC-3'

[0147] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne SUS (D296Q / H531L / D569Q).

[0148] (4) pET-28a(+)- Ne Using SUS(D296Q / H531L / D569Q) as the template plasmid, and E610N_F / E610N_R as the primer pair, a fourth round of site-directed mutagenesis was performed. The primers were designed as follows:

[0149] E610N_F:5'-CATTCCTCTGACCACGAAaatCAGGAACAAATCCACCG -3'

[0150] E610N_R: 5'-CGGTGGATTTGTTCCTGattTTCGTGGTCAGAGGAATG-3'

[0151] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne SUS (D296Q / H531L / D569Q / E610N).

[0152] (5) pET-28a(+)- Ne Using SUS(D296Q / H531L / D569Q / E610N) as the template plasmid, and E663N_F / E663N_R as the primer pair, a fifth round of site-directed mutagenesis was performed. The primers were designed as follows:

[0153] E663N_F:5'-CGTGCAGCCGGCCTGTTTaatGCATTTGGTCTGACCATC -3'

[0154] E663N_R:5'-GATGGTCAGACCAAATGCattAAACAGCGCCGGCTGCACG-3'

[0155] The remaining steps are as follows (1), obtaining the mutant plasmid pET-28a(+)- Ne The nucleotide sequence of the mutant SUS (D296Q / H531L / D569Q / E610N / E663N) and the mutated sucrose synthase (D296Q / H531L / D569Q / E610N / E663N) are shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10.

[0156] 3. Preparation of sucrose synthase (D296Q / H531L / D569Q / E610N / E663N) mutant enzyme solution

[0157] The mutant plasmid pET-28a(+)- Ne Recombinant engineered E. coli BL21(DE3) was obtained by transforming SUS (D296Q / H531L / D569Q / E610N / E663N) into E. coli BL21(DE3). Ne SUS (D296Q / H531L / D569Q / E610N / E663N). Recombinant engineered E. coli BL21(DE3)- NeSingle colonies of SUS (D296Q / H531L / D569Q / E610N / E663N) were inoculated into 4.0 mL of LB broth containing Kana resistance and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculum to 50 mL of LB broth containing Kana resistance and cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM–1 mM and cultured at 20–37°C with shaking for 12.0–16.0 h. After induction, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.2). The cells were then sonicated in an ice bath, and the lysate was centrifuged. The supernatant was collected to obtain the sucrose synthase. Ne Crude enzyme solution of SUS (D296Q / H531L / D569Q / E610N / E663N) mutant.

[0158] 4. Sucrose synthase Ne Enzyme activity assay of SUS (D296Q / H531L / D569Q / E610N / E663N) mutant

[0159] Using 26 mg sucrose and 21 mg ADP as substrates, 0.5 mg of crude enzyme solution of the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant prepared in Part 3 was added, bringing the total volume to 1 ml. The pH was adjusted to 5.0, and the reaction was carried out at 75℃ and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity.

[0160] 5. Sucrose synthase Ne Preparation of SUS (D296Q / H531L / D569Q / E610N / E663N) mutants; optimization of conversion rates at different reaction pH in RD.

[0161] Using rebaudioside A 2.0 g as substrate, sucrose 0.7 g, ADP 44.0 mg, and UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) 5.0 mg were added, along with 5.0 mg of crude enzyme solution of the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant prepared in Part 3, for a total volume of 10.0 ml. The reaction was carried out at 75 °C and 200 rpm. After the reaction, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid phase sample for HPLC analysis to calculate the conversion rate.

[0162] At a temperature of 75℃, pH gradients of 4.0, 5.0, 6.0, 7.0, and 8.0 were set, and the conversion rates at different pH values ​​were measured. The results are shown in the table below:

[0163]

[0164] 6. Preparation of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutants and optimization of conversion rates at different reaction temperatures under optimal pH (pH 6.0) in RD.

[0165] Using rebaudioside A 2.0 g as substrate, sucrose 0.7 g, ADP 44.0 mg, and UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) 5.0 mg, and then adding the crude enzyme solution of the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant prepared in Part 3, the total volume was 10.0 ml, and the reaction was carried out at 200 rpm. After the reaction, the reaction solution was diluted 100 times with methanol, filtered through a 0.22 μm microfiltration membrane, and used as a liquid phase sample for HPLC analysis to calculate the conversion rate.

[0166] Under pH 7.0 conditions, temperature gradients of 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃ were set, and the conversion rates at different temperatures were measured. The results are shown in the table below:

[0167]

[0168] 7. Optimization of ADP substrate feed rate for preparation of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutants

[0169] Prepare separate solutions of ADP 22 mg, ADP 11 mg, ADP 7 mg, and ADP 6 mg, sucrose 0.7 g, and RA 2 g, respectively, and stir until completely dissolved. Add 5.0 mg of crude enzyme solution of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) and 5.0 mg of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant, with a total volume of 10.0 ml for each solution, and adjust the pH to 7. Incubate the solutions at 65℃ and 200 rpm for 6 h. After the reaction, dilute the reaction solution 100 times with methanol, filter through a 0.22 μm microfiltration membrane, and use as a liquid phase sample for HPLC analysis.

[0170] The conversion rate of rebaudioside D was 76.2% when ADP was 22 mg; 75.7% when ADP was 11 mg; 74.2% when ADP was 7 mg; and 64.2% when ADP was 6 mg.

[0171] 8. Optimization of dosage of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant

[0172] Prepare a solution of 2.0 g rebaudioside A, 0.7 g sucrose, and 7.0 mg ADP, stirring until completely dissolved. Add 5.0 mg of crude enzyme solution of UDP-glucosyltransferase mutant (K290E / Q342W / F354G / L367I) (GenBank accession number: XP_015629141.1) and 3.0 mg, 4.0 mg, and 5.0 mg of NeSUS sucrose synthase mutant (D296Q / H531L / D569Q / E610N / E663N), respectively, with a total volume of 10.0 ml for each sample. Adjust the pH to 7. Incubate the reaction at 65℃ and 200 rpm for 6 h. After the reaction, dilute the reaction solution 100 times with methanol, filter through a 0.22 μm microfiltration membrane, and use as a liquid phase sample for HPLC analysis.

[0173] When the dosage of the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant was 3.0 mg, the conversion rate of rebaudioside D was 68.8%; when the dosage of the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant was 4.0 mg, the conversion rate of rebaudioside D was 75.9%; and when the dosage of the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant was 5.0 mg, the conversion rate of rebaudioside D was 75.2%.

[0174] 9. pH stability analysis of sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutants

[0175] pH stability: Sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant enzyme solutions were placed in buffer solutions ranging from pH 5.5 to 9.5 and incubated at 4°C for 24 hours. The remaining enzyme activity was then measured. The results showed that the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutants maintained over 70% enzyme activity within the pH range of 5-7, while the wild-type maintained the same level only within the pH range of 5.5-6.5.

[0176] The wild type is only stable at pH 5.5-6.5, while the sucrose synthase NeSUS (D296Q / H531L / D569Q / E610N / E663N) mutant has a wider stability range and is more suitable for neutral reaction systems in some industrial scenarios (such as some buffers whose natural pH is close to 7.0).

[0177] Comparative Example 1: Enzyme Activity Assay of Sucrose Synthase Parent

[0178] 1. Preparation of sucrose synthase parent plasmids

[0179] derived from Nitrosaminoglycans Nitrosomonas europaea Sucrose synthase Ne After codon optimization of SUS (Genbank accession number CAD85125.1), the required gene fragment was synthesized and ligated into the pET28a vector, with Nde I and Xho I restriction enzyme sites at both ends, to obtain the sucrose synthase parent pET-28a(+)- Ne The SUS plasmid was then transformed into E. coli DH5α. After Kana screening, colonies were selected and sequenced to determine the nucleotide sequence of the cloned sucrose synthase parent as shown in SEQ ID NO: 1, and its amino acid sequence as shown in SEQ ID NO: 2.

[0180] 2. Preparation of parent enzyme solution of sucrose synthase

[0181] Recombinant engineered E. coli BL21(DE3) was obtained by transforming the parental plasmid into E. coli BL21(DE3). Ne SUS. Recombinant engineered E. coli BL21(DE3)- NeSUS was inoculated at a ratio of 1% into 4.0 mL of liquid LB medium and cultured overnight at 37°C with shaking (200 rpm). The overnight culture was then transferred at a 1% inoculation rate to a large volume of liquid LB medium and cultured at 37°C with shaking (200 rpm) until the OD600 value reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM–1 mM and cultured at 20–37°C with shaking for 12.0–16.0 h. After induction, the cells were collected by centrifugation and resuspended in 50 mM phosphate buffer (pH 7.2). The cells were then sonicated in an ice bath, and the lysate was centrifuged. The supernatant was collected to obtain the crude enzyme solution containing the parental mutant of sucrose synthase.

[0182] 3. Determination of enzyme activity of sucrose synthase parents

[0183] Using 26 mg sucrose and 21 mg ADP as substrates, 0.5 mg of crude sucrose synthase NeSUS prepared in Part 2 was added, bringing the total volume to 1 ml. The pH was adjusted to 5, and the reaction was carried out at 75 °C and 200 rpm for 30 min. Samples were then taken for HPLC analysis to calculate enzyme activity.

[0184] 4. Determination of the reaction temperature of the sucrose synthase parent.

[0185]

[0186] 5. The enzyme activity test results of the sucrose synthase mutant and its parent are as follows:

[0187]

[0188] As shown in the table above, the mutant enzyme activity of the present invention is increased by 12% to 38% compared with the wild type, and the conversion rate of rebaudioside D is as high as 84.2% (in Example 3, under the conditions of 60℃, pH 6.0, and 6h), and the stability is good in the range of pH 5.0-7.0 and 55-75℃.

[0189] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A sucrose synthase mutant, characterized in that, The nucleotide sequences of the sucrose synthase mutants are shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:

9.

2. The sucrose synthase mutant according to claim 1, characterized in that, The amino acid sequence of the sucrose synthase mutant is shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:

10.

3. A method for preparing rebaudioside D from a sucrose synthase mutant, characterized in that, Using the sucrose synthase mutant and β-1,2-glucosidase as catalysts, and rebaudioside A, sucrose, and ADP as substrates, a reaction is carried out to generate rebaudioside D.

4. The method for preparing rebaudioside D from the sucrose synthase mutant according to claim 3, characterized in that, The reaction temperature is controlled at 55℃-80℃, the pH is 4.0-8.0, and the reaction time is 0.5h-18h.

5. The method for preparing rebaudioside D according to claim 3 or the sucrose synthase mutant, characterized in that, The reaction temperature is controlled at 55℃-75℃, the pH is 5.0-7.0, and the reaction time is 4h-18h.

6. The method for preparing rebaudioside D from the sucrose synthase mutant according to claim 3, characterized in that, The reaction temperature was controlled at 65℃-70℃, the pH was 6.0, and the reaction time was 6 hours.

7. The method for preparing rebaudioside D from the sucrose synthase mutant according to any one of claims 4-6, characterized in that, The reaction is carried out in a solution containing the following components, wherein: The initial reaction concentration of rebaudioside A is 160-300 g / L. The initial reaction concentration of sucrose is 60-80 g / L. The initial reaction concentration of ADP is 0.4-4.4 g / L. The initial reaction concentration of β-1,2-glucosidase was 0.4-0.6 g / L. The crude enzyme solution of the sucrose synthase mutant according to any one of claims 1-2 has a total protein concentration of 0.3-0.5 g / L in the reaction system. The remainder is solvent.

8. A method for preparing rebaudioside D from a sucrose synthase mutant according to any one of claims 5-6, characterized in that, The reaction is carried out in a solution containing the following components, wherein: The initial reaction concentration of rebaudioside A was 200 g / L. The initial reaction concentration of sucrose was 70 g / L. The initial reaction concentration of ADP was 0.7 g / L. The initial reaction concentration of β-1,2-glucosidase was 0.5 g / L. The crude enzyme solution of the sucrose synthase mutant according to any one of claims 1-2 has a total protein concentration of 0.5 g / L in the reaction system. The remainder is pure water; The pH was adjusted to 6.0, and the reaction was carried out at 60°C for 18 hours to catalyze the formation of rebaudioside D.

Images