Glycosyltransferase UGT94B1 mutant with improved thermal stability and activity

Abstract

The invention discloses a glycosyl transferase UGT94B1 mutant with improved thermal stability and activity, and belongs to the technical field of biological catalytic synthesis. Through rational design, the 11th site, the 96th site, the 219th site, the 281th site and the 327th site of glycosyl transferase are mutated to obtain the combined mutant UGT94B1M3, compared with a starting enzyme, the half-life period at 40 DEG C is prolonged from 0.43 h to 8.88 h, the thermal stability is improved by 20.47 times, and the enzyme activity is improved by 1.45 times. According to the invention, the mutant is expressed in escherichia coli, and a cascade reaction is constructed with sucrose synthase, so that the Reb D conversion rate reaches 84.67%, and an efficient new way is provided for production of rebaudioside D.

Description

Technical Field

[0001] This invention relates to a glycosyltransferase UGT94B1 mutant with enhanced thermal stability and activity, belonging to the field of biocatalytic synthesis technology. Background Technology

[0002] Steviol glycosides are a class of natural sweeteners extracted from stevia, with over 60 types identified to date, differing mainly in the number and position of glucose units attached to their side chains. Among them, the rare rebaudioside D (Reb D) is 250 to 400 times sweeter than sucrose and has a much smaller bitter residue than rebaudioside A (Reb A), making it particularly favored in the food and beverage industry, helping to balance the dual demands of flavor and health. Unfortunately, Reb D is present in extremely low amounts in plants (only 0.1% to 0.5% of the dry leaf weight), making traditional extraction methods both expensive and inefficient. Therefore, enzymatic conversion of the abundant Reb A to Reb D using uridine diphosphate glycosyltransferases (UGTs) has become a promising alternative. Several UGTs capable of catalyzing this conversion have been reported, including YojK, OsUGT91C1, UGT94B1, and UGTSL2, whose performance has been further improved through directed evolution or rational design. For example, Shoji et al. enhanced the Reb A activity of UGT91D2 by modifying it, while the inventors' team previously obtained the mutant UGT94B1-I146C / Y164G through rational design of UGT94B1, which improved catalytic efficiency by 5.3 times. Despite the improved catalytic activity, the mutant exhibited poor thermostability in cascade reactions, leading to reduced efficiency and limiting the enzyme's scalability. Therefore, improving the thermostability of UGT94B1-I146C / Y164G is a crucial step towards achieving efficient and sustainable biocatalytic production of Reb D. Summary of the Invention

[0003] Based on UGT94B1-I146C / Y164G, this invention successfully obtained a combined mutant with improved activity and thermal stability. This mutant can efficiently catalyze the generation of Reb A from Reb D, thereby meeting the requirements of industrial production.

[0004] This invention provides a glycosyltransferase mutant, specifically the previously constructed glycosyltransferase UGT94B1-I146C / Y164G (abbreviated as UGT94B1). M0 Based on this, one or more amino acids at positions 11, 96, 219, 281, and 327 are mutated.

[0005] In one embodiment, glycosyltransferase UGT94B1M0 The amino acid sequence is shown in SEQ ID NO.3.

[0006] In one implementation, the mutation is any one of (1) to (5): (1) Mutate the 11th alanine to leucine; (2) Mutate the threonine at position 96 to methionine; (3) Mutate threonine at position 219 to lysine; (4) Mutate threonine at position 281 to asparagine; (5) Mutate serine at position 327 to proline.

[0007] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.3, with alanine at position 11 mutated to leucine and threonine at position 219 mutated to lysine, and named UGT94B1. M1 .

[0008] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.3, with alanine at position 11 mutated to leucine, threonine at position 96 mutated to methionine, threonine at position 219 mutated to lysine, and threonine at position 281 mutated to asparagine, named UGT94B1. M2 .

[0009] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.3, with alanine at position 11 mutated to leucine, threonine at position 96 mutated to methionine, threonine at position 219 mutated to lysine, threonine at position 281 mutated to asparagine, and serine at position 327 mutated to proline, named UGT94B1. M3 .

[0010] In one embodiment, the glycosyltransferase mutant UGT94B1 M3 The amino acid sequence is shown in SEQ ID NO.1.

[0011] The present invention also provides a gene encoding the glycosyltransferase mutant.

[0012] The present invention also provides an expression vector carrying the gene.

[0013] The present invention also provides recombinant microbial cells expressing the glycosyltransferase mutant.

[0014] In one embodiment, the recombinant microorganism uses Escherichia coli as a host.

[0015] In one embodiment, the recombinant microorganism uses Escherichia coli BL21(DE3) as the host and pET-21b(+) as the vector to express the glycosyltransferase mutant.

[0016] In one embodiment, the recombinant bacteria also co-express the glycosyltransferase mutant with sucrose synthase.

[0017] In one embodiment, the recombinant bacteria express the glycosyltransferase mutant using pET-21b(+) as a vector and express sucrose synthase using pACYCDuet-1 as a vector.

[0018] In one embodiment, the amino acid sequence of the sucrose synthase is shown in SEQ ID NO.5.

[0019] The present invention also provides a biocatalyst containing the glycosyltransferase mutant and sucrose synthase.

[0020] In one embodiment, the biocatalyst contains the active enzyme protein of the glycosyltransferase mutant and sucrose synthase, or contains recombinant microbial cells expressing the glycosyltransferase mutant and sucrose synthase; or contains cell lysate of the recombinant microbial cells.

[0021] In one embodiment, the cell lysis buffer is the supernatant obtained by culturing the recombinant bacteria in a culture medium for a period of time, inducing the expression of the glycosyltransferase mutant with an inducer, and then lysing the cells.

[0022] This invention also provides a method for the catalytic synthesis of Reb D, using Reb A as a substrate and a biocatalyst for the catalytic reaction.

[0023] In one embodiment, the method involves culturing the recombinant bacteria in LB medium for 8-12 hours to obtain a seed culture, then transferring it to 2×YT medium and culturing at 35-40°C until OD reaches the target value. 600 The temperature was lowered from 0.6-0.8℃ to 16-22℃, and IPTG was used to induce cell production for 10-15 hours. Cells were collected from the fermentation broth, and the supernatant was collected as cell lysis buffer after cell disruption using a high-pressure homogenizer. This supernatant was then used for the biosynthesis of Reb D.

[0024] In one embodiment, the reaction system contains (by final concentration): 10-100 mM Reb A, 0-3 mM UDP, 200-800 mM sucrose, 10-1000 mmol / L KPi buffer, and 10-1000 mmol / L NaCl, and the reaction is carried out at 20-60°C for at least 4 h.

[0025] In one embodiment, the sucrose concentration is 400-800 mmol / L.

[0026] In one embodiment, the catalytic reaction is carried out at 30-40°C; particularly at 35-40°C or 37±0.5°C.

[0027] In one implementation, the reaction takes at least 40 minutes.

[0028] In one implementation, the reaction takes 1 hour.

[0029] This invention also protects the above-mentioned glycosyltransferase mutant or the above-mentioned gene UGT94B1. M3 The above expression vector, the above microbial cells, the above recombinant bacteria, or the above method may be used in the preparation of products containing Reb D.

[0030] Beneficial effects: (1) This invention uses the previously constructed glycosyltransferase UGT94B1 mutant as a template for protein engineering modification, and successfully obtains a highly efficient mutant UGT94B1 with improved thermostability and activity of the enzyme. M3 Compared to the starting enzyme, this mutant exhibits significantly enhanced thermostability compared to UGT94B1. M0 It increased by 20.47 times, and the catalytic efficiency also increased by 1.45 times.

[0031] (2) This invention constructs a recombinant strain capable of co-expressing the ginseng-derived glycosyltransferase mutant UGT94B1. M3 Sucrose synthase from Arabidopsis thaliana At SuSy. By inducing recombinant bacterial expression and preparing cell lysates, the lysates were used to catalyze the synthesis of Reb D from Reb A. After optimizing the cascade reaction system conditions, the mutant UGT94B1... M3 The reaction rate of RebA compared to the starting enzyme UGT94B1 M0 Significant improvement.

[0032] The glycosyltransferase mutant constructed in this invention can effectively promote the industrialization of Reb D biosynthesis, reduce the amount of enzyme used, and lower production costs. Attached Figure Description

[0033] Figure 1 UGT94B1 in Example 5 M0 And the results of the optimal temperature test for the combined mutants.

[0034] Figure 2 UGT94B1 in Example 6 M0 And the results of half-life tests for combined mutants.

[0035] Figure 3 This is the effect of pH on the synthesis of Reb D from Reb A in the enzyme cascade reaction in Example 9.

[0036] Figure 4 This illustrates the effect of temperature on the synthesis of Reb D from Reb A in the enzyme cascade reaction, as shown in Example 10.

[0037] Figure 5 The effect of sucrose concentration on the synthesis of Reb D from Reb A catalyzed by the enzyme cascade reaction in Example 11.

[0038] Figure 6 The effect of UDP concentration on the synthesis of Reb D from Reb A in the enzyme cascade reaction is shown in Example 12.

[0039] Figure 7 The effect of DMSO concentration on the synthesis of Reb D from Reb A in the enzyme cascade reaction is shown in Example 13.

[0040] Figure 8 The effect of cell cleavage concentration on the synthesis of Reb D from Reb A catalyzed by the enzyme cascade reaction in Example 14.

[0041] Figure 9 The effect of Reb A concentration on the synthesis of Reb D from Reb A in the enzyme cascade reaction is shown in Example 15.

[0042] Figure 10 This illustrates the effect of reaction time on the synthesis of Reb D from Reb A via the enzyme cascade reaction in Example 16. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0044] Unless otherwise specified, all reagents and materials used in the following examples are commercially available or can be prepared by known methods. Reb glycoside A (Reb A) and Reb glycoside D (Reb D) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0045] (a) Culture medium: LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder.

[0046] 2×YT liquid medium: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl.

[0047] (II) Testing methods: Detection of Reb D: Reb D was dissolved in DMSO to prepare a 5 mM mother liquor. The mother liquor was then diluted with methanol to prepare solutions of various concentrations: 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, and 1.75 mM. After filtration through a 0.22 μm filter membrane, the peak areas were analyzed by UPLC. A Reb D standard concentration curve was fitted using Origin 8.0 software, with Reb D concentration on the x-axis and peak area on the y-axis. The yield of Reb D was calculated based on the standard curve. Yield = Actual Reb D yield / Theoretical Reb D yield × 100%.

[0048] Example 1: Glycosyltransferase UGT94B1 M0 Construction of mutants and strains 1. Construction of single mutants The recombinant plasmid pET-21b(+)-UGT94B1 containing the UGT94B1-I146C / Y164G gene shown in SEQ ID NO.4 was used. M0 Using A11L-1 and A11L-2 as templates, full plasmid PCR was performed (primer sequences are shown in Table 1) to construct the recombinant plasmid pET-21b(+)-UGT94B1 carrying the mutant A11L. M0 -A11L.

[0049] The resulting plasmid pET-21b(+)-UGT94B1 M0 -A11L was sequenced, identified, and transformed into E. coli. E. coli In BL21(DE3), positive colonies containing the target gene were screened to obtain recombinant strains. E. coli BL21(DE3) pET-21b(+)-UGT94B1 M0 -A11L.

[0050] Table 1 Primer names and primer sequences

[0051] The recombinant plasmid pET-21b(+)-UGT94B1 containing the following mutant was constructed using the same strategy described above. M0 -T96M、pET-21b(+)-UGT94B1 M0 -T219K、pET-21b(+)-UGT94B1 M0 -T281N and pET-21b(+)-UGT94B1 M0 -S327P, and the relevant primer sequences are shown in Table 1. The constructed recombinant plasmids were then transformed into *E. coli*. E. coliRecombinant bacteria were constructed from BL21(DE3) competent cells as follows: E. coli BL21(DE3) pET-21b(+)-UGT94B1 M0 -T96M, E. coli BL21(DE3) pET-21b(+)-UGT94B1 M0 -T219K, E. coli BL21(DE3) pET-21b(+)-UGT94B1 M0 -T281N and E. coli BL21(DE3) pET-21b(+)-UGT94B1 M0 -S327P.

[0052] 2. Construction of combined mutants With UGT94B1 M0 As a template, first import T m The two single-point mutations, A11L and T219K, which significantly improved the results, yielded UGT94B1. M0 -A11L / T219K (hereinafter referred to as UGT94B1) M1 ).

[0053] With UGT94B1 M1 Using this as a template, we introduce activity enhancement, T m The two single-point mutations T96M and T281N, which were improved by a small amount, yielded UGT94B1. M0 -A11L / T96M / T219K / T281N (hereinafter referred to as UGT94B1) M3 ).

[0054] With UGT94B1 M2 Using the template, the mutation S327P was introduced to obtain UGT94B1. M0 -A11L / T96M / T219K / T281N / S327P (hereinafter referred to as UGT94B1) M3 ).

[0055] The primers used for mutation are shown in Table 1. The recombinant plasmids containing the combined mutants were transformed into *E. coli*. E. coli Recombinant bacteria containing combined mutants were obtained from BL21(DE3) competent cells: E. coli BL2l(DE3)-pET-21b(+)-UGT94B1 M1 , E. coli BL2l (DE3)-pET-21b(+)-UGT94B1 M2 and E. coliBL2l(DE3)-pET-21b(+)-UGT94B1 M3 .

[0056] Example 2: Induction of recombinant strain expression and purification of target protein The recombinant strain constructed in Example 1 E. coli BL21(DE3) pET-21b(+)-UGT94B1 M0 The mutant strains were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37 ℃ and 220 rpm for 8–12 h. 5 mL of the seed culture was then transferred to 500 mL of 2×YT medium containing 100 μg / mL ampicillin and cultured at 37 ℃ and 115 rpm until OD (dose retardation). 600 The target cytokine concentration was 0.6-0.8. Cells were induced with 0.1 mM isopropyl-β-thiogalactoside (IPTG) and cultured at 18 °C for 12 h. Cells were collected by centrifugation and resuspended in buffer A (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerol). Cells were homogenized using an autoclave, and the supernatant (cell lysis buffer) was collected by centrifugation. The supernatant was purified using a Ni-NTA column: after loading, contaminating proteins were washed with 50 mL buffer B (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 15 mmol / L imidazole, 10% glycerol); the target protein was eluted with 4 mL buffer C (50 mmol / L Tris-HCl pH 8.0, 250 mmol / L NaCl, 250 mmol / L imidazole, 10% glycerol). The protein concentration and purity of the eluent were determined using a Nanodrop 2000. The high-imidazolium fraction was replaced with buffer D (50 mmol / L Tris-HCl pH 8.0, 150 mmol / L NaCl) using Amicon® Ultra ultrafiltration tubes. The purified protein was ready for immediate use or could be stored at -80°C.

[0057] Example 3: UGT94B1 M0 Enzyme activity assay The purified glycosyltransferase UGT94B1 obtained in Example 2 M0 Its mutants were used in the glycosylation reaction. The glycosylation reaction system (200 μL) is shown in Table 2.

[0058] Table 2 Glycosylation Reaction System

[0059] The system described in Table 2 was reacted at 35℃ and 800 rpm for 10 min, and then the reaction was terminated by heating at 95℃. 400 μL of methanol was added, and the mixture was centrifuged at 20000×g for 5 min. The supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by UPLC (method as described in previous studies). All experiments were repeated three times, and the Reb D content in the reaction system was quantified using a standard curve.

[0060] UPLC was performed using a CORTECS C18 1.6 μm column (2.1 × 50 mm). The HPLC conditions were: A - acetonitrile, B - ultrapure water; flow rate 0.3 mL / min; column temperature 35 ℃; UV detection wavelength 210 nm; detection program: 0-1 min 15% A, 7 min 50% A, 8-9 min 15% A.

[0061] The Reb D content was determined by liquid chromatography using UGT94B1. M0 The transformation effect was used as a standard to calculate the relative enzyme activity of the mutants. The results are shown in Table 3. The mutants A11L, T281N, and UGT94B1... M0 The enzyme activity compared to UGT94B1 M0 There were significant improvements, with increases of 46%, 50%, and 45% respectively.

[0062] Example 4: UGT94B1 M0 Test of the denaturation temperature of its mutants UGT94B1 was prepared according to the method in Example 2. M0 The purified enzyme, including its mutants, was used to test the enzyme's denaturation temperature. The specific steps are as follows: UGT94B1 was measured using Nano-DSC. M0 and the thermal denaturation temperature of its mutants ( T m The enzyme and buffer system used in the test (based on final concentration) was: 2 mg / mL LUGT94B1 M0 Or its mutant, 50 mmol / L Tris-HCl pH 8.0 buffer, 150 mmol / L NaCl. Degassed for 10 min before the experiment. Baseline correction was repeated three times using the desalting buffer as a reference. After equilibration at a constant pressure of 3 atm for 10 min, the temperature was increased from 10℃ to 70℃ at a scan rate of 1℃ / min. Measurements were performed under these conditions. T m Changes in value.

[0063] Relative enzyme activity is defined as: under the same detection conditions, the enzyme activity of the mutant relative to UGT94B1 is measured and calculated. M0 Percentage of enzyme activity. Following the method in Example 3, UGT94B1...M0 The enzyme activity of UGT94B1 and its mutants was detected. M0 The enzyme activity was set at 100%, and the relative enzyme activity data of other mutants were calculated. As shown in Table 3, compared with UGT94B1... M0 All mutants T m It shows a significant improvement, and the enzyme activity of most mutants is also enhanced.

[0064] Table 3. Changes in relative enzyme activity and denaturation temperature of mutants

[0065] Δ T m : with UGT94B1 M0 Difference in thermal denaturation temperature Example 5: UGT94B1 M0 Test of the optimal reaction temperature of its combined mutants UGT94B1 was prepared according to the method in Example 2. M0 The purified enzymes, including their combined mutants, were used to test the optimal temperature of the enzymes. The specific procedures are as follows: The reaction system (200 μL) contained 0.02 mg / mL enzyme, 5 mM Reb A, 10 mM UDPG, and 50 mM Tris-HCl (pH 8.0). Reactions were carried out at 25–50 °C and 800 rpm for 10 min, respectively, followed by termination at 95 °C. 400 μL of methanol was added, and the mixture was centrifuged at 20000 × g for 5 min. The supernatant was filtered through a 0.22 μm organic filter and analyzed by UPLC. The amount of glycosylated products generated in the reaction system was detected to calculate UGT94B1. M0 The enzyme activity of its mutants was measured, and the highest enzyme activity was counted as 100% relative enzyme activity.

[0066] The results are as follows Figure 1 As shown, UGT94B1 M0 With UGT94B1 M1 The optimal temperature is 40℃, while UGT94B1 M2 and UGT94B1 M3 The optimal temperature compared to UGT94B1 M0 The temperature was increased by 5℃, to 45℃. This result indicates that the final mutant significantly improved the optimal reaction temperature of the enzyme.

[0067] Example 6: UGT94E13 M0 Testing of the half-life of its combined mutants UGT94E13 was prepared according to the method in Example 2. M0The purified enzymes, including their combined mutants, were tested for their half-life. The specific steps are as follows: To determine UGT94B1 M0 Half-life of proteins and their mutants t 1 / 2 ), UGT94E13 M0 The enzyme activity of the mutant (or its combination mutants, 2 mg / mL) was measured at 40°C after incubation at 0.15, 0.5, 0.75, 1, 2, 4, 6, 8, and 10 h. The reaction system and processing method were the same as those for UGT94B1 in Example 3. M0 Enzyme activity assay. The enzyme activity of untreated samples is defined as 100%. The activity of samples after incubation at 40 °C is expressed as the percentage of residual enzyme activity (%) relative to the initial activity. Samples at 40 °C... t 1 / 2 The calculation formula is as follows: t 1 / 2 =ln2 / k d (k) d (Represents the deactivation rate constant).

[0068] like Figure 2 As shown, mutant UGT94E13 M3 The half-life at 40℃ is significantly longer, at 8.88 h, compared to UGT94B1. M0 The half-life of (UGT94B1-I146C / Y164G) is increased by 20.47 times.

[0069] Example 7: Construction of recombinant plasmids and recombinant strains of glycosyltransferase and sucrose synthase According to sucrose synthase from Arabidopsis thaliana published by Genbank. At The SuSy amino acid sequence (amino acid sequence as shown in SEQ ID NO. 5) was optimized for codon preference in *E. coli* and synthesized by Yixin Biotechnology Co., Ltd. (nucleotide sequence as shown in SEQ ID NO. 6), and then ligated to the NcoI / NflII site of the vector pACYCDuet-1 to obtain the recombinant plasmid pACYCDuet-1- At SuSy identified the obtained plasmid by sequencing, and then combined it with the recombinant plasmid pET-21b(+)-UGT94B1 constructed in Example 1. M3 Co-transformation into Escherichia coli E. co1i Recombinant strains were obtained by screening for correct colonies in BL21(DE3) competent cells on LB solid medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. E. coli BL21(DE3) pET-21b(+)-UGT94B1M3 pACYCDuet-1- At SuSy.

[0070] Example 8: Co-expression of glycosyltransferase UGT94B1 M3 and sucrose synthase At Preparation of cell lysate from SuSy recombinant strain The recombinant strain constructed in Example 7 E. coli BL21(DE3) pET-21b(+)-UGT94B1 M3 pACYCDuet-1- At SuSy was the fermentation strain. Cells were cultured according to the induction expression method of the recombinant strain in Example 2, and cell lysates were prepared. The crude protein concentration of the cell lysates was measured using a Nano-Drop 2000 UV-Vis spectrophotometer. The prepared cell lysates were aliquoted and stored at -80 °C, or used directly for enzyme-catalyzed reactions.

[0071] Example 9: Effect of pH on the synthesis of Reb D from Reb A by the enzyme cascade reaction Cell lysis buffer was prepared according to the method in Example 8 to study the effects of various factors on the efficiency of the enzyme cascade reaction. A 1 mL cascade reaction system was constructed, containing 30 mg / mL cell lysis buffer, 1 mM UDP, 20 mM Reb A, 200 mM sucrose, and 10% DMSO. The buffer solutions for the reaction system were either phosphate buffer (pH 5.5–8.0) containing 100 mM NaCl or Tris-HCl (pH 7.5–9.0) containing 100 mM NaCl. The reaction system was incubated at 35 °C and 220 rpm for 30 min. After the reaction, the sample was inactivated at 95 °C for 5 min, diluted 20-fold with methanol, centrifuged at 20000 × g for 5 min, and the supernatant was filtered through a 0.22 μm organic filter before UPLC analysis. The results showed ( Figure 3 The highest Reb D yield (64.50%) was observed when the buffer solution was 100 mmol / L KPi pH 6.5 (containing 100 mmol / L NaCl).

[0072] Example 10: Effect of temperature on the synthesis of Reb D from Reb A by the enzyme cascade reaction The reaction system was prepared according to the method of Example 9, except that the buffer solution was 100 mmol / L KPi pH 6.5 (containing 100 mmol / L NaCl). Reactions were carried out at 20–50 °C (temperature intervals of 5 °C), with other conditions remaining unchanged. The effect of temperature on the cascade reaction was tested according to the standard reaction system of Example 9. The results showed ( Figure 4When the temperature is 35 degrees Celsius o At C, the RebD yield is the highest, at 75.76%.

[0073] Example 11: Effect of sucrose concentration on the synthesis of Reb D from Reb A catalyzed by the enzyme cascade reaction The reaction system was prepared according to the method in Example 9, except that the buffer solution was 100 mmol / L KPi pH 6.5 (containing 100 mmol / L NaCl), and the sucrose solution was adjusted to 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, and 1000 mM, respectively. Other conditions remained unchanged, and the effect of sucrose concentration on the enzyme cascade reaction was tested according to the standard reaction system of Example 9. The results showed ( Figure 5 When the sucrose concentration was 500 mM, the Reb D yield was the highest, at 74.02%.

[0074] Example 12: Effect of UDP concentration on the synthesis of Reb D from Reb A catalyzed by the enzyme cascade reaction The reaction system was prepared according to the method in Example 9, except that the buffer solution was 100 mmol / L KPi pH 6.5 (containing 100 mmol / L NaCl), the sucrose concentration was 500 mM, and the UDP concentrations were set to 0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, and 5 mM, respectively. Other conditions remained unchanged, and the effect of different UDP concentrations on the enzyme cascade reaction was tested according to the standard reaction system of Example 9. The results showed ( Figure 6 When the UDP concentration was 1.5 mM, the Reb D yield was the highest, at 69.16%.

[0075] Example 13: Effect of DMSO concentration on the synthesis of Reb D from Reb A catalyzed by the enzyme cascade reaction The effect of DMSO concentration on the synthesis of Reb D from Reb A catalyzed by the enzyme cascade reaction was determined. The reaction system was prepared according to the method in Example 9, except that the buffer solution was 100 mmol / L KPi pH 6.5 (containing 100 mmol / L NaCl), the sucrose concentration was 500 mM, and the UDP concentration was 1.5 mM. Different DMSO concentration gradients (0%, 5%, 10%, 15%, 20%, 25%) were set. Other conditions remained unchanged, and the effect of different DMSO concentrations on the enzyme cascade reaction was tested using the standard reaction system of Example 9. The results showed ( Figure 7 When the DMSO concentration was 0%, the Reb D yield was the highest, at 68.48%.

[0076] Example 14: Effect of cell lysate concentration on the synthesis of Reb D from Reb A catalyzed by the enzyme cascade reaction. Based on the optimized parameters from Examples 9-13, the following reaction system was prepared (based on final concentration): 100 mmol / L KPi pH 6.5 (containing 100 mmol / L NaCl), 500 mmol / L sucrose, and 1.5 mmol / L UDP, with different cell lysis buffer concentration gradients (10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, and 50 mg / mL). The reaction was carried out at 35 ℃ and 220 rpm for 40 min to test the effect of different cell lysis buffer concentrations on the enzyme cascade reaction. The results showed ( Figure 8 When the cell lysis buffer concentration was 40 mg / mL, the Reb D yield reached 95.14%.

[0077] Example 15: Effect of Reb A concentration on the synthesis of Reb D from Reb A catalyzed by the enzyme cascade reaction The reaction system was prepared according to the method in Example 14, except that the cell lysis buffer concentration was 40 mg / mL, and different Reb A concentration gradients were set (10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L). The reaction was carried out at 35 ℃ and 220 rpm for 30 min, and the effect of different Reb A concentrations on the enzyme cascade reaction was tested. The results showed ( Figure 9 When the Reb A concentration is less than 30 mmol / L, the Reb D yield can exceed 70%. To obtain a relatively high Reb D yield, we selected 40 mM Reb A as the substrate concentration for the final scale-up.

[0078] Example 16: Effect of reaction time on the synthesis of Reb D from Reb A by the enzyme cascade reaction To prepare Reb D (at a final concentration), a 10 mL reaction system was prepared. The reaction system (at a final concentration, containing): 100 mmol / L KPi (containing 100 mmol / L NaCl, pH 6.5), 40 mg / mL cell lysis buffer, 500 mM sucrose, 40 mM RebA, and 1.5 mM UDP. The reaction was carried out at 35℃ and 250 rpm for 4 h. Samples were taken every 0.5 h to determine the yield of Reb D. Each reaction was performed in triplicate. The results are as follows: Figure 10 As shown, the yield of Reb D reached 84.67% after 1 h of reaction, but the yield of Reb D did not increase significantly as the reaction proceeded. This may be because the large amount of Reb D precipitated in the system affected the subsequent reaction.

[0079] Comparative Example 1: The specific implementation method is the same as in Examples 1 and 2, except that the mutant shown in Table 4 was also constructed. The relative enzyme activity was detected by the method in Example 3, and the thermal stability was detected by the method in Example 4. The results are shown in Table 4.

[0080] Table 4 Effects of different mutants

[0081] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A glycosyltransferase mutant, characterized in that, Based on the parent, one or more amino acids at positions 11, 96, 219, 281, and 327 are mutated; the amino acid sequence of the parent is shown in SEQ ID NO.

3.

2. The glycosyltransferase mutant according to claim 1, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.3, it has any of the mutations described in (a) to (c): (a) Mutate alanine at position 11 to leucine and threonine at position 219 to lysine; (b) Mutate alanine at position 11 to leucine, threonine at position 96 to methionine, threonine at position 219 to lysine, and threonine at position 281 to asparagine; (c) Mutate alanine at position 11 to leucine, threonine at position 96 to methionine, threonine at position 219 to lysine, threonine at position 281 to asparagine, and serine at position 327 to proline.

3. A gene encoding the glycosyltransferase mutant of claim 1 or 2.

4. An expression vector carrying the gene of claim 3.

5. Recombinant microbial cells of the glycosyltransferase mutant according to claim 1 or 2.

6. Recombinant Escherichia coli, characterized in that, The glycosyltransferase mutant was expressed using Escherichia coli BL21(DE3) as the host and pET-21b(+) as the vector.

7. The recombinant Escherichia coli according to claim 6, characterized in that, The glycosyltransferase mutant was also co-expressed with sucrose synthase; the amino acid sequence of the sucrose synthase is shown in SEQ ID NO.

5.

8. A biocatalyst containing the glycosyltransferase mutant of claim 1 or 2 and the sucrose synthase shown in SEQ ID NO.

5.

9. A method for catalytic synthesis of Reb D, characterized in that, Using Reb A as a substrate, a catalytic reaction is carried out using the biocatalyst described in claim 8. Optionally, the sucrose concentration in the reaction system is ≥400 mM; 0.5~3.0 mM UDP is added to the reaction system; and the catalytic reaction is carried out at 30~40°C.

10. The use of the glycosyltransferase mutant of claim 1 or 2, or the gene of claim 3, or the recombinant microbial cell of claim 5, or the recombinant Escherichia coli of any one of claims 6-7, or the biocatalyst of claim 8, or the method of claim 9 in the preparation of a product containing rebaudioside D.

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