Sucrose phosphorylase mutants and their use in the preparation of aa-2g

Abstract

The application provides a sucrose phosphorylase mutant and application thereof in preparation of AA-2G. The application forms a recombinant genetically engineered bacterium by mutation and modification of sucrose phosphorylase and a promoter T7 in an expression vector, and applies the recombinant genetically engineered bacterium to catalytic synthesis of AA-2G, so that the synthesis yield of AA-2G is improved, the conversion rate is improved, and the impurity content is reduced. The method is green and efficient, and is suitable for industrial production of AA-2G.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a sucrose phosphorylase mutant and its application in the preparation of 2-O-α-D-glucopyranosyl-L-ascorbic acid. Background Technology

[0002] 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a common L-ascorbic acid (L-AA or VC) derivative, formed by the substitution of the hydroxyl group at the C2 position of L-AA with a pyranoside by glycosyltransferases. It exists as a white crystalline powder and has a higher solubility than vitamin C. Due to the instability of L-AA, AA-2G often appears in daily life as a substitute for L-AA. Compared to traditional L-AA, AA-2G exhibits better stability in the presence of oxygen, heat, and metal ions.

[0003] When used, AA-2G is hydrolyzed into L-AA by α-glucosidase in the skin cell membrane, allowing it to exert its specific effects efficiently and continuously. As a recognized whitening additive, it can directly remove melanin from the epidermis by reverse-reducing melanin. AA-2G inhibits the formation of melanin in the skin, is stable and easily absorbed, and is therefore often used as a main ingredient in skincare products for whitening, spot removal, anti-oxidation, and anti-aging. In terms of health benefits, AA-2G can lower cholesterol levels, prevent viral and bacterial infections, enhance immunity, fight cancer, prevent scurvy, and is a main ingredient in anti-allergy and anti-urinary tract infection drugs.

[0004] Currently, the main methods for synthesizing AA-2G are chemical and enzymatic methods. Chemical synthesis involves complex steps, poor regioselectivity, numerous byproducts, and difficulties in separation and purification. It also incurs solvent residues and environmental pressures, making it difficult to meet high-purity requirements. Enzymatic conversion, catalyzed by glycosyltransferases, offers advantages such as mild reaction conditions, high selectivity, high product purity, and environmental safety, making it the mainstream industrial route. The enzymatic synthesis of AA-2G using genetically engineered bacteria expressing glycosyltransferases can be rapidly scaled up for industrial production. Furthermore, it leverages the rapid reproduction and simple cultivation of microorganisms, resulting in low pollutant emissions during production, aligning with the direction of green and low-carbon industrial development. However, existing methods for catalytic synthesis of AA-2G still have certain drawbacks, such as low product yield and conversion rate, and high impurity content. Therefore, there is an urgent need for a green, low-cost, and industrially scaled-up method for preparing AA-2G that achieves high yield, high conversion rate, and low impurities. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a sucrose phosphorylase mutant and its use in the preparation of AA-2G. By mutating the sucrose phosphorylase and the promoter T7 in the expression vector, a recombinant genetically engineered bacterium is formed and applied to the enzymatic synthesis of AA-2G, thereby increasing the yield of AA-2G, improving the conversion rate, and reducing the impurity content. The method is green, efficient, and suitable for the industrial production of AA-2G.

[0006] On the one hand, the present invention provides a sucrose phosphorylase mutant, which is based on the sequence of sucrose phosphorylase as shown in SEQ ID No. 3 and has a mutation site, which includes any one or more of A272V, P434S, E63D, I189L, P480S, T288S, D168A, D447E, T455S or G459S.

[0007] To optimize the performance of sucrose phosphorylase, the inventors modified the enzyme through site mutations. By screening for single-point mutations and combined site mutations, they discovered that when sucrose phosphorylase was mutated in one or more of the following forms: A272V, P434S, E63D, I189L, P480S, T288S, D168A, D447E, T455S, or G459S, the concentration and conversion rate of the catalytic product AA-2G increased, and the impurity content decreased. This may be because the change in amino acid composition after mutation optimizes the internal interactions of the enzyme molecule, making the overall spatial conformation more stable, thus enabling the catalytic process to continue efficiently. In addition, it reduces side reactions, improves the selectivity of the target catalysis, and makes it more specific to the target substrate.

[0008] Furthermore, the mutant is obtained by mutating three sites, P434S, I189L and P480S, in the amino acid sequence shown in SEQ ID No. 3.

[0009] During the screening of single-point mutations and site combination mutations of sucrose phosphorylase, the inventors discovered that mutants obtained by mutating at three sites (P434S, I189L, and P480S) in the amino acid sequence shown in SEQ ID No. 3 exhibit superior catalytic effects on the product, achieving a product concentration of 186.0 g / L, a product conversion rate of 55%, and an impurity content of 0.04%. Compared to single-point mutations, two-site mutations, and other three-point mutations, this mutant demonstrates the best catalytic effect. Compared to mutants with more than three sites, the catalytic effect is comparable. This indicates that under the conditions of this site mutation combination, the optimal catalytic effect (increasing product yield and conversion rate, and reducing impurity content) can be maintained, and the mutant preparation process is the simplest.

[0010] Furthermore, the amino acid sequence of the mutant is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.4.

[0011] In another aspect, the present invention provides a recombinant gene expression vector comprising the nucleotide sequence of the above-mentioned mutant.

[0012] The nucleotide sequence of the aforementioned sucrose phosphorylase mutant was introduced into a recombinant gene expression vector, enabling the recombinant expression vector to express the sucrose phosphorylase mutant. On the one hand, this eliminates the need for enzyme isolation and purification, allowing for the acquisition of the catalytic unit containing the target enzyme, thus reducing production costs. On the other hand, it improves enzyme stability and lifespan, and allows for flexible regulation of enzyme expression and activity. The expression vector itself contains controllable expression regulatory elements, such as promoters, which can precisely control the timing and amount of enzyme expression to meet the needs of different production rhythms. Furthermore, the expression level of the target enzyme can be increased by modifying and optimizing the codons through vector modification, thereby further improving catalytic efficiency.

[0013] Furthermore, the promoter of the recombinant gene expression vector is a T7 promoter mutant, which is obtained by any one or more mutations of T1A, A2T, T4A, T4C or G7A in the sequence shown in the nucleotide sequence TAATACGACTCACTATAG.

[0014] Promoters can alter transcription initiation through site mutations. Appropriate promoter mutations can significantly improve the expression efficiency of exogenous genes while addressing the problems of unstable expression and easy silencing associated with conventional promoters. Mutation screening can yield promoter variants with varying activities, enabling precise control over the expression level of target genes and adapting to different expression needs. For high-volume expression of target proteins, potent synergistic mutations can be selected to increase yield, offering greater flexibility than wild-type promoters with fixed strength. Screening for single-point and combined site mutations of the T7 promoter in gene expression vectors has revealed that promoter mutants obtained by any one or more mutations in the T7 promoter (T1A, A2T, T4A, T4C, or G7A) exhibit superior catalytic activity in recombinant gene expression vectors containing sucrose phosphorylase mutant genes, increasing catalytic product yield and conversion rate while reducing impurity content.

[0015] Furthermore, the T7 promoter mutant is obtained by mutating T4C on the sequence shown in the nucleotide sequence TAATACGACTCACTATAG.

[0016] During the screening of single point mutations and combined site mutations of the T7 promoter, the T7-T4C promoter mutant showed superior catalytic effect compared with other single point mutants. Furthermore, further mutations did not significantly change the catalytic effect, indicating that the T7-T4C promoter mutant in the recombinant gene expression vector ensures the best catalytic effect while simplifying the mutation process.

[0017] In another aspect, the present invention provides a recombinant genetically engineered bacterium, wherein the recombinant genetically engineered bacterium contains the above-mentioned recombinant gene expression vector.

[0018] By culturing recombinant genetically engineered bacteria carrying recombinant gene expression vectors, catalytic units containing the target enzyme can be obtained, eliminating the high costs of purification processes. Simultaneously, the expression vector can stably replicate and passage within host cells, allowing for long-term amplification and use of the engineered bacteria after a single construction, making it suitable for large-scale industrial production. Furthermore, the expressed enzyme is protected within the host cell, reducing the impact of external environmental factors (temperature fluctuations, pH changes, heavy metal impurities) on enzyme activity, thus significantly improving enzyme stability. Recombinant genetically engineered bacteria containing the aforementioned recombinant gene expression vector initiate transcription with a promoter mutant, translating and synthesizing a sucrose phosphorylase mutant, thereby achieving the expression of the sucrose phosphorylation mutant in the genetically engineered bacteria.

[0019] Furthermore, the host bacteria of the recombinant genetically engineered bacteria are one or more of Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum.

[0020] Preferably, the host bacterium of the recombinant genetically engineered bacteria is Escherichia coli.

[0021] The above recombinant expression vectors were introduced into Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum to construct recombinant genetically engineered bacteria, which were then applied to the preparation of AA-2G. It was found that sucrose phosphorylase mutants could all be expressed and catalyze L-AA glycosylation to produce AA-2G. When the host bacterium was Escherichia coli, the catalytic product yield was the highest, the conversion rate was the largest, and the impurity content was the lowest.

[0022] Preferably, the host bacteria of the recombinant genetically engineered bacteria are E. coli BL21(DE3).

[0023] In another aspect, the present invention provides the use of the above-mentioned sucrose phosphorylase mutant, recombinant gene expression vector or recombinant genetically engineered bacteria in the preparation of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

[0024] In another aspect, the present invention provides the use of the above-mentioned sucrose phosphorylase mutant, recombinant gene expression vector or recombinant genetically engineered bacteria in the preparation of a catalytic agent that improves the conversion rate and yield of 2-O-α-D-glucopyranosyl-L-ascorbic acid and reduces the impurity content.

[0025] The sucrose phosphorylase mutant, recombinant expression vector, and recombinant genetically engineered bacteria provided by this invention have the following beneficial effects: The sucrose phosphorylase mutant provided by this invention can be used for the enzyme-catalyzed preparation of AA-2G, which can improve the yield and conversion rate of the catalytic product and produce no impurities (3-O-α-glucosyl-L-ascorbic acid).

[0026] The recombinant gene expression vector containing the sucrose phosphorylase gene and the recombinant genetically engineered bacteria containing the expression vector provided by this invention can be used for enzyme-catalyzed preparation of AA-2G, which can improve the yield and conversion rate of the catalytic product and produce no impurities (3-O-α-glucosyl-L-ascorbic acid). Attached Figure Description

[0027] Figure 1 A schematic diagram of the preparation of AA-2G catalyzed by sucrose phosphorylase; Figure 2 The high-performance liquid chromatogram of the substrate L-ascorbic acid standard is shown below. Figure 3 The high-performance liquid chromatogram of the product AA-2G standard is shown below. Figure 4 The standard curve of the substrate L-ascorbic acid; Figure 5 The standard curve spectrum of the product AA-2G; Figure 6 Agarose gel electrophoresis patterns of PCR products obtained by mutation of sucrose phosphorylase SPase at the T7 promoter; (a) Lane M is the marker; lanes 1-14 are respectively: T7, T7-T1A, T7-T1C, T7-T1G, T7-A2T, T7-A3T, T7-T4A, T7-T4C, T7-T4G, T7-A5C, T7-A5T, T7-C6G, T7-C6T, T7-G7A; (b) Lane M is the marker; lanes 1-5 are respectively: T7-A8T, T7-A12T, T7-C13G, T7-T16A, T7-A17T; Figure 7SDS-PAGE analysis of wet cells induced by mutant expression of sucrose phosphorylase SPase at the T7 promoter; (a) Lane M is the marker; lanes 1-10 are: T7, T7-T1A, T7-T1C, T7-T1G, T7-A2T, T7-A3T, T7-T4A, T7-T4C, T7-T4G, T7-A5C; (b) Lane M is the marker; lanes 1-9 are: T7-A5T, T7-C6G, T7-C6T, T7-G7A, T7-A8T, T7-A12T, T7-C13G, T7-T16A, T7-A17T; Figure 8 High-performance liquid chromatography (HPLC) chromatogram of the reaction solution catalyzed by sucrose phosphorylase Spase; Figure 9 High-performance liquid chromatogram of the reaction solution catalyzed by the sucrose phosphorylase mutant Spase-I189L / P434S / P480S-T4C; Figure 10 This is the high-performance liquid chromatogram of the purified AA-2G sample. Figure 11 This is a graph showing the change in product conversion rate over reaction time. Figure 12 This is a map of plasmids for recombinant gene expression vectors. Detailed Implementation

[0028] To describe the present invention more specifically, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are merely illustrative of how the present invention is implemented and do not limit the specific scope of the present invention. The scope of the present invention is defined in the claims.

[0029] Example 1: E. coli Construction of BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C The codon-encoding gene for sucrose phosphorylase SPase (nucleotide sequence shown in SEQ ID NO.1) derived from *Bifidobacterium longum* underwent codon optimization. The nucleotide sequence of the codon-optimized SPase gene is shown in SEQ ID NO.2, and the corresponding amino acid sequence of sucrose phosphorylase SPase is shown in SEQ ID NO.3. This gene was synthesized by Hangzhou Qingke Biotechnology Co., Ltd. The codon-optimized SPase gene nucleotide sequence (SEQ ID NO.2) was then artificially synthesized and inserted into pET28a. Nco I and Xho The recombinant plasmid pET28a-SPase was obtained between I and II.

[0030] Take 5 μL of the synthesized recombinant expression plasmid pET28a-SPase and add it to 50 μL. E. coli In BL21(DE3) competent cells, gently tap the tube wall to mix, and place on ice for 30 min. Heat shock in a 42 ℃ water bath for 45 s, then immediately place on ice for 2 min. Add 1 mL of LB liquid medium (yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, distilled water as solvent, pH 7.0-7.5) to the tube, and incubate at 37 ℃ on a shaker for 1 h. Centrifuge the culture at 4500 rpm for 4 min, and collect 800 μL of supernatant. Resuspend the cells in the remaining medium, and spread 100 μL onto LB solid medium containing 100 μg / mL kanamycin. Incubate overnight at 37 ℃ for 12-14 h to obtain the genetically engineered bacteria. E. coli BL21(DE3) / pET28a-SPase, and extract plasmid pET28a-SPase.

[0031] Using plasmid pET28a-SPase as an amplification template, a mutant was constructed using reverse PCR. The PCR product was then supplemented with... Dpn I restriction endonuclease 1 µL, reacted at 37 ℃ for 1 h to remove methylated template. Then, the PCR product was recovered and purified using a DNA gel purification kit and stored at -20 ℃ for later use. The recombinant plasmid obtained from the mutation was transformed into the host bacterium *Escherichia coli*. E. coli BL21(DE3) yielded genetically engineered bacteria. E. coli BL21(DE3) / pET28a-SPase-I189L / P434S / P480S was extracted, and the plasmid pET28a-SPase-I189L / P434S / P480S was extracted.

[0032] Using plasmid pET28a-SPase-I189L / P434S / P480S as an amplification template, a vector for the promoter mutant T7-T4C was constructed using reverse PCR. The PCR product was then supplemented with... Dpn I. 1 µL of restriction endonuclease was added, and the reaction was carried out at 37 °C for 1 h to remove the methylated template. The PCR product was then purified using a DNA gel purification kit and stored at -20 °C for later use. The target fragment obtained from the mutation was directly transformed into the host bacterium *Escherichia coli*. E. coli BL21(DE3): Take 50 μL E. coliBL21(DE3) competent cells were thawed on ice, and 5 μL of the mutant PCR product was added. The cells were then incubated on ice for 30 min. After the ice bath, the competent cells were heat-shocked at 42 °C for 45 s, and then immediately placed on ice for 3-5 min. 1 mL of antibiotic-free LB medium was added, and the cells were incubated at 37 °C and 200 rpm for 1 h. After incubation, the culture was centrifuged at 4 °C and 4500 rpm for 5 min, and 900 μL of the supernatant was discarded. The remaining cells were thoroughly resuspended, and 100-150 μL of the bacterial culture was spread onto LB agar plates containing kanamycin resistance. The cells were incubated upside down at 37 °C for 12-16 h to obtain the genetically engineered *Escherichia coli*. E. coli BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C.

[0033] Example 2: Induced expression of sucrose phosphorylase SPase and its mutants The genetically engineered bacteria constructed in Example 1 E. coli BL21(DE3) / pET28a-SPase and E. coli BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C were inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured at 37℃ for 12 h to obtain seed culture. The seed culture was then inoculated into fresh LB liquid medium containing 100 μg / mL kanamycin at a volume concentration of 2% and cultured at 37℃ until the OD600 reached 0.5–0.7. Then, isopropyl-β-D-thiogalactoside (IPTG, Huibai Reagent, reagent grade) was added to a final concentration of 0.2 mM and induced at 24℃ for 12 h to obtain induction culture. The culture was then centrifuged at 4℃ and 10,000 rpm for 10 min, the supernatant was discarded, and the wet cells were collected.

[0034] Add an appropriate amount of buffer solution to the above wet bacterial cells at a ratio of 1 g wet bacterial cells to 5 mL citrate-sodium citrate buffer (pH 5.4) (Maclean, reagent grade). Sonicate at 125 W for 15 min (2 s working, 6 s rest). Centrifuge the lysate at 4℃ and 10000 rpm for 10 min. Repeat the centrifugation three times to obtain the supernatant crude enzyme solution.

[0035] Example 3: Construction of the catalytic reaction system of sucrose phosphorylase SPase and its mutants Using the crude enzyme solution of sucrose phosphorylase SPase and its mutant prepared by the method described in Example 2 as a catalyst, a 30 mL reaction system was constructed, comprising L-AA (Maclean, reagent grade), sucrose (Maclean, reagent grade), crude enzyme solution, and citrate buffer (Maclean, reagent grade). The reaction was carried out in a reactor at 35 °C and pH 5.4. The amounts of each component added are shown in Table 1. Sucrose was added every 1 h, and 1.5 M was added at 8 h. Sucrose was then added continuously until 16 h, after which the reaction continued without adding more sucrose until 27 h. Figure 1 A schematic diagram of the preparation of AA-2G catalyzed by sucrose phosphorylase.

[0036] Table 1. Reaction system for the preparation of AA-2G catalyzed by sucrose phosphorylase

[0037] Example 4: Liquid Chromatographic Analysis of AA-2G Preparation Catalyzed by Sucrose Phosphorylase SPase 1. Treatment of reaction solution The catalytic reaction system was constructed using the method in Example 3. 1 mL of sample was centrifuged at 10,000 rpm for 2 min. The supernatant was diluted 100 times with the mobile phase, filtered through a membrane, and then used for sample loading.

[0038] 2. High Performance Liquid Chromatography (HPLC) Analysis Method The substrate L-AA and product AA-2G can be detected by high-performance liquid chromatography (HPLC). The HPLC conditions are as follows: column: ACE Excel 5 C18-PFP; mobile phase: 6.86 g KH2PO4, 5 mL methanol, diluted to 1 L with pure water, pH adjusted to 2.0 with phosphoric acid; wavelength: 238 nm; flow rate: 0.7 mL / min; column temperature: 25 ℃; injection volume: 10 μL; hold time: 15 min. The HPLC chromatogram of the substrate L-AA standard is shown below. Figure 2 As shown, the elution time was 6.260 min, and the liquid chromatography chromatogram of the product AA-2G standard is as follows. Figure 3 As shown, the peak elution time is 6.982 min.

[0039] Vitamin C standards with different concentration gradients (0.02 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, and 0.3 g / L) were prepared. Peak areas were obtained using this chromatographic method. A vitamin C standard curve was plotted with vitamin C concentration on the x-axis and peak area on the y-axis, as shown below. Figure 4 As shown, the formula for obtaining the standard curve is y = 74892050.9x - 238981.02. The conversion rate is calculated based on the obtained substrate standard curve.

[0040] AA-2G standards with different concentration gradients (0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1.0 g / L) were prepared. Peak areas were obtained using this chromatographic method. A standard curve was plotted with AA-2G concentration on the x-axis and peak area on the y-axis, as shown below. Figure 5 As shown, the formula for obtaining the standard curve is y = 27581824.5x - 60622.3. The concentrations of the product and impurities are calculated based on the obtained product standard curve.

[0041] Example 5: Single-point mutation of sucrose phosphorylase SPase The sucrose phosphorylase (SPase) gene was sequence aligned, homology modeled, and molecular docking analyzed to identify 42 amino acid sites for single-point mutation. The amino acid sequences of the SPase mutants are as follows: (1) Replace the glutamic acid at position 63 of the amino acid sequence shown in SEQ ID No.3 with aspartic acid and proline, respectively; (2) Replace the aspartic acid at position 69 of the amino acid sequence shown in SEQ ID No.3 with glycine; (3) Replace the asparagine at position 79 of the amino acid sequence shown in SEQ ID No.3 with glycine; (4) Replace the aspartic acid at position 127 of the amino acid sequence shown in SEQ ID No.3 with glutamic acid; (5) Replace the aspartic acid at position 168 of the amino acid sequence shown in SEQ ID No.3 with alanine; (6) Replace the lysine at position 169 of the amino acid sequence shown in SEQ ID No.3 with arginine, histidine, or glutamic acid, respectively; (7) Replace the serine at position 187 of the amino acid sequence shown in SEQ ID No.3 with arginine; (8) Replace the isoleucine at position 189 of the amino acid sequence shown in SEQ ID No.3 with leucine; (9) Replace the alanine at position 272 of the amino acid sequence shown in SEQ ID No.3 with valine; (10) Replace histidine at position 273 of the amino acid sequence shown in SEQ ID No.3 with arginine; (11) Replace the glutamic acid at position 276 of the amino acid sequence shown in SEQ ID No.3 with aspartic acid; (12) Replace the threonine at position 288 of the amino acid sequence shown in SEQ ID No.3 with serine; (13) Replace the valine at position 341 of the amino acid sequence shown in SEQ ID No.3 with isoleucine and alanine, respectively; (14) Replace the alanine at position 427 of the amino acid sequence shown in SEQ ID No.3 with serine; (15) Replace the proline at position 434 of the amino acid sequence shown in SEQ ID No.3 with serine and threonine, respectively; (16) Replace the aspartic acid at position 437 of the amino acid sequence shown in SEQ ID No.3 with glutamic acid; (17) Replace the glycine at position 438 of the amino acid sequence shown in SEQ ID No.3 with serine; (18) Replace the glutamic acid at position 439 of the amino acid sequence shown in SEQ ID No.3 with aspartic acid; (19) Replace the phenylalanine at position 440 of the amino acid sequence shown in SEQ ID No.3 with serine; (20) Replace the aspartic acid at position 445 of the amino acid sequence shown in SEQ ID No.3 with glutamic acid; (21) Replace the glycine at position 446 of the amino acid sequence shown in SEQ ID No.3 with serine; (22) Replace the aspartic acid at position 447 of the amino acid sequence shown in SEQ ID No.3 with glutamic acid; (23) Replace the threonine at position 448 of the amino acid sequence shown in SEQ ID No.3 with serine; (24) Replace the threonine at position 455 of the amino acid sequence shown in SEQ ID No.3 with serine; (25) Replace the alanine at position 456 of the amino acid sequence shown in SEQ ID No.3 with valine; (26) Replace the alanine at position 457 of the amino acid sequence shown in SEQ ID No.3 with valine; (27) Replace the aspartic acid at position 458 of the amino acid sequence shown in SEQ ID No.3 with glutamic acid; (28) Replace the glycine at position 459 of the amino acid sequence shown in SEQ ID No.3 with serine; (29) Replace the threonine at position 460 of the amino acid sequence shown in SEQ ID No.3 with alanine and serine, respectively; (30) Replace serine at position 461 of the amino acid sequence shown in SEQ ID No.3 with proline; (31) Replace the proline at position 469 of the amino acid sequence shown in SEQ ID No.3 with serine; (32) Replace the proline at position 480 of the amino acid sequence shown in SEQ ID No.3 with serine; (33) Replace the arginine at position 496 of the amino acid sequence shown in SEQ ID No.3 with alanine, histidine, or glutamic acid, respectively; (34) Replace the proline at position 502 of the amino acid sequence shown in SEQ ID No.3 with serine; (35) Replace the leucine at position 343 of the amino acid sequence shown in SEQ ID No.3 with isoleucine, proline, or asparagine, respectively; (36) Replace the 91st tryptophan in the amino acid sequence shown in SEQ ID No.3 with alanine; (37) Replace proline at position 134 of the amino acid sequence shown in SEQ ID No.3 with cysteine; (38) Replace valine at position 154 of the amino acid sequence shown in SEQ ID No.3 with threonine; (39) Replace histidine at position 185 of the amino acid sequence shown in SEQ ID No.3 with glycine; (40) Replace the asparagine at position 424 of the amino acid sequence shown in SEQ ID No.3 with leucine; (41) Replace the asparagine at position 396 of the amino acid sequence shown in SEQ ID No.3 with lysine; (42) Replace the asparagine at position 397 of the amino acid sequence shown in SEQ ID No.3 with phenylalanine; Using the plasmid pET28a-SPase prepared in Example 1 as an amplification template, a mutant was constructed using reverse PCR technology. The primers used are shown in Table 2, and the PCR amplification system is shown in Table 3.

[0042] Table 2 Primers for single-point mutation of sucrose phosphorylase SPase

[0043]

[0044]

[0045] Table 3 PCR amplification reaction system

[0046] The PCR reaction procedure was as follows: pre-denaturation at 95℃ for 5 min; then, a cycle of denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and holding at 72℃ for 1 min was performed, repeated 30 times; finally, holding at 72℃ for 5 min. The PCR products were detected by 0.8% agarose gel electrophoresis, and the bands matched the theoretical values ​​of the plasmid. 1 µL of Dpn I restriction endonuclease was added to the PCR products, and the reaction was carried out at 37℃ for 1 h to remove the methylated template. The PCR products were then purified using a DNA gel purification kit and stored at -20℃ for later use.

[0047] The recombinant plasmid obtained by mutation was transformed into the host bacterium *Escherichia coli* using the method for preparing genetically engineered bacteria described in Example 1. E. coli BL21(DE3) (without promoter mutation) yielded the E. coli BL21(DE3) / pET28a-SPase mutant. The mutants were named accordingly; for example, when proline at position 434 of the amino acid sequence was replaced with serine, the genetically engineered bacterium was named... E. coli BL21(DE3) / pET28a-SPase-P434S.

[0048] The engineered bacteria were induced to express sucrose phosphorylase and its mutants using the method in Example 2. The expression effect was verified by SDS-PAGE electrophoresis. The molecular weight of SPase and its mutants was approximately 56 kDa. The protein bands expressed by each mutant strain were significantly thickened and located at approximately 56 kDa, which was the correct size.

[0049] To verify the activity of different mutants, wet cells of SPase and its mutants were prepared using the method in Example 2, and crude enzyme solutions were obtained by disruption. The SPase and its mutants were then subjected to catalytic reactions using the method in Example 3. The catalytic results of sucrose phosphorylase and its mutants were analyzed by liquid chromatography using the analytical method in Example 4. The catalytic effect was compared by comparing the consumption of substrate L-AA and the generation of product AA-2G in the catalytic reaction solution over 4 hours. The results are shown in Table 4. The experimental results showed that the concentration of the SPase enzyme catalyzed product was 101.5 g / L, the product conversion rate was 30.0%, and the impurity content was 0.30%. Chromatographic analysis excluded fructose and glucose, suggesting that the impurities were disaccharidated or polysaccharidated L-ascorbic acid derivatives and AA glycoside isomers bound at different sites. Based on the single peak and fixed retention time of the impurities, and considering the catalytic mechanism of sucrose phosphorylase, the impurities were verified by standard analysis to be 3-O-α-glucosyl-L-ascorbic acid. SPase-A272V, SPase-P434S, SPase-E63D, SPase I189L, SPase-P480S, SPase-T288S, SPase-D168A, SPase-D447E, SPase-T455S, and SPase-G459S all improved the yield and conversion rate of AA-2G. Except for SPase-D168A, all of them reduced the impurity content of the product. Among them, SPase-P480S showed the best overall performance. After 4 hours of reaction, the product conversion rate reached 43.7%, the yield reached 147.8 g / L, and the impurity (3-O-α-glucosyl-L-ascorbic acid) content was 0.11%.

[0050] Table 4. Catalytic results of SPase and its mutants detected by liquid phase.

[0051]

[0052] Using the plasmid pET28a-SPase-P480S prepared in Example 5 as an amplification template, combined mutant strains were constructed using reverse PCR technology according to the method described in Example 5. The list of combined mutant strains is shown in Table 5. Wet cells of SPase and its mutants were prepared using the method in Example 2 and crushed to obtain crude enzyme solution. The crude enzyme solution was used to catalyze the reaction using the method in Example 3. The catalytic results of the cells were analyzed by liquid chromatography using the method in Example 4. The catalytic effect and impurity content of each mutant were compared by comparing the conversion rate of product AA-2G in the catalytic reaction solution after 4 h. The experimental results are shown in Table 5. The results showed that the catalytic product concentration and conversion rate of the double-mutant mutants were higher than those of SPase, while the impurity content was lower. Among them, SPase-I189L / P480S and SPase-P434S / P480S showed the best performance, with product yields of 154.2 g / L and 140.7 g / L, product conversion rates of 45.6% and 41.6%, and impurity contents of 0.06% and 0.07%, respectively. Further mutations showed that these mutants had better catalytic product concentration, conversion rate, and impurity content than SPase. Among them, SPase-I189L / P434S / P480S showed the best performance, with a product concentration of 186.0 g / L, a product conversion rate of 55.0%, and an impurity content of 0.04%. Further mutations did not show significant differences in catalytic effect.

[0053] Table 5. Results of liquid phase detection of SPase and its mutant engineered bacteria.

[0054] Example 7: Single-point mutation of the T7 promoter The T7 promoter single-point mutation scheme is as follows: (1) Replace the first base T in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with A; (2) Replace the first base T in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with C; (3) Replace the first base T in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with G; (4) Replace the second codon A in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with T; (5) Replace the third base A in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with T; (6) Replace the fourth base T in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with A; (7) Replace the fourth base T in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with C; (8) Replace the fourth base T in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with G; (9) Replace the fifth base A in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with C; (10) Replace the fifth base A in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with T; (11) Replace the 6th base C in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with G; (12) Replace the 6th base C in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with T; (13) Replace the 7th base G in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with A; (14) Replace the 8th base A in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with T; (15) Replace the 12th base A in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with T; (16) Replace the 13th base C in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with G; (17) Replace the 16th base T in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with A; (18) Replace the 17th base A in the T7 promoter (T7 promoter sequence: TAATACGACTCACTATAG) with T.

[0055] Using plasmid pET28a-SPase-I189L / P434S / P480S as amplification template, vectors with different promoter sequences were constructed using reverse PCR. The T7 promoter sequence is TAATACGACTCACTATAG (SEQ ID NO.150). The primers are shown in Table 6, and the PCR amplification system is shown in Table 7.

[0056] PCR reaction program: pre-denaturation: 95 ℃, 5 min; complete denaturation: 95 ℃, 15 s; annealing: 59 ℃, 15 s; extension: 72 ℃, 90 s; 30 cycles; second extension: 72 ℃, 5 min; cool to 4 ℃ and incubate.

[0057] Table 6 Primers for T7 promoter mutation

[0058] Table 7 PCR system for full plasmid amplification

[0059] After the PCR reaction, an appropriate amount of the PCR product was taken for agarose gel electrophoresis verification. After electrophoresis, bright bands were observed under UV light between 5000-8000 bp, consistent with the theoretical values ​​for the plasmid. The agarose gel electrophoresis results are as follows: Figure 6 As shown. Then, add to the PCR product. Dpn Incubate with 1 µL of restriction endonuclease at 37 °C for 1 h to remove methylated template. Then, PCR products are purified using a DNA gel extraction kit. Store at -20 °C for later use.

[0060] The target fragment obtained from the mutation was directly transformed into the host bacterium *Escherichia coli*. E. coli BL21(DE3): Take 50 μL E. coli BL21(DE3) competent cells were thawed on ice, and 5 μL of the mutant PCR product was added. The cells were then incubated on ice for 30 min. After the ice bath, the competent cells were heat-shocked at 42 °C for 45 s, and immediately placed on ice for 3-5 min. 1 mL of antibiotic-free LB medium was added, and the cells were incubated at 37 °C and 200 rpm for 1 h. After incubation, the culture was centrifuged at 4 °C and 4500 rpm for 5 min, and 900 μL of the supernatant was discarded. The remaining cells were thoroughly resuspended, and 100-150 μL of the bacterial culture was spread onto LB agar plates containing Kan antibiotics. The cells were incubated upside down at 37 °C for 12-16 h to obtain the genetically engineered *E. coli* strain. E. coli BL21(DE3) / pET28a-SPase-I189L / P434S / P480S mutants, for example, when the T7 promoter base is mutated to T1A, the genetically engineered bacteria are named... E. coli BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T1A.

[0061] Wet bacterial cells of engineered bacteria were obtained by inducing expression under different promoters using the method in Example 2. The expression effect was verified by SDS-PAGE electrophoresis. The electrophoresis results are as follows: Figure 7 As shown, the molecular weight of SPase protein is approximately 56 kDa. The protein bands expressed by each strain are significantly thickened and positioned around 56 kDa, which is the correct size.

[0062] To verify the activity of different mutants, engineered bacterial wet cells were obtained by inducing expression under different promoters using the method in Example 2, and crude enzyme solutions were obtained by disruption. The T7 promoter and its base substitutions were catalyzed using the method in Example 3. The reaction solution after 4 h was analyzed by high-performance liquid chromatography using the method described in Example 4. The results are shown in Table 8. Compared with the T7 promoter, the base substitutions of T1A, A2T, T4A, T4C, and G7A led to an increase in the conversion rate of product AA-2G and a decrease in impurity content to 0, which were confirmed as beneficial mutations. Among them, T4C had the highest conversion rate.

[0063] Table 8. Catalytic results of engineered bacteria with T7 promoter and base substitutions detected by liquid phase.

[0064] Note: * indicates a beneficial mutation. by E. coli BL21(DE3) / pET28a-SPase and mutant strains E. coli Taking BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C as an example, the plasmid map of pET28a-SPase-I189L / P434S / P480S-T4C is as follows: Figure 12 As shown. High-performance liquid chromatography (HPLC) analysis was performed using the method described in Example 4. The results are as follows. Figure 8 As shown, when the strain E. coli When the BL21(DE3) / pET28a-Spase catalyzed reaction was analyzed by liquid chromatography, a small impurity peak was found at 10.699 min. However, when a mutant strain was used... E. coli When BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C is catalyzed, the results are as follows: Figure 9 As shown, no impurity peaks were observed, indicating that the mutant strain has higher selectivity, thus ruling out the possibility of impurity formation due to the generation of other isomers during the catalytic reaction.

[0065] Example 8: Multisite mutations in the T7 promoter Prepared in Example 7 E. coliBL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C was further screened for promoter multi-site mutations, and the promoter mutation screening groups are shown in Table 9. Experimental results showed that after multi-site mutations, the product concentration and conversion rate of the T7 promoter were significantly higher than before the mutation. T7-T4C and promoter mutants further mutated based on this promoter showed comparable catalytic effects.

[0066] Table 9. Catalytic results of engineered bacteria with T7 promoter and multi-site base substitutions detected by liquid phase.

[0067] Example 9: High-density fermentation, enzyme solution preparation, and catalytic effect verification of sucrose phosphorylase SPase and mutant SPase-I189L / P434S / P480S-T4C 1. Seed culture strain E. coli BL21(DE3) / pET28a-SPase and strain E. coli Remove BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C from a -80 ℃ freezer. Streak a glycerol tube onto a slant containing 100 μg / mL kanamycin. Incubate at 37 ℃ for 13-15 h. Add 6 mL of sterile water to the incubated slant and use an inoculation loop to scrape the bacterial culture from the slant into the sterile water. Inoculate 1% of the culture into LB medium and incubate at 37 ℃ and 220 rpm for 10-11 h. At this point, OD... 600 Around 5.0.

[0068] LB liquid medium consists of: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, distilled water as solvent, pH 7.0-7.5. LB solid medium is LB liquid medium with 20 g / L agar added.

[0069] 2. High-density fermentation Prepare 5 L of fermentation medium, as shown in Table 10. Prepare 1 L of fed-batch medium, as shown in Table 11. Sterilize both fermentation and fed-batch media at 121 °C for 30 min. After sterilization, when the temperature drops to 60 °C, add kanamycin at 100 μg / mL to the fermentation medium. When the temperature drops to 37 °C, inoculate the seed culture into the fermentation medium at a 2.0% inoculum rate (100 mL / 5 L) and incubate at 37 °C. Aeration should begin at 1 vvm and increase to 1.2 vvm after 3 h. Maintain tank pressure at 0.05-0.06 MPa. Stirring speed should be 500-700 rpm. Dissolved oxygen should be maintained at a minimum of 20%. Dissolved oxygen decreases rapidly after 2 h of fermentation and then rises rapidly after about 5 h; at this point, fed-batch medium should be added to maintain dissolved oxygen at approximately 30%. The pH may rise slowly in the early stages of fermentation, then decrease after feeding. The pH can then be adjusted with ammonia to maintain a range of 6.7-6.9. After feeding begins, the temperature should be slowly lowered to 30-33 °C for 1-2 hours. When OD... 600 When the temperature reaches 20-25℃, cool it down to 24℃. Add 0.2 mM IPTG and induce for 14-16 hours before removing from the container.

[0070] Table 10 Fermentation medium formulation (5 L)

[0071] Table 11 Feeding medium formulation (1 L)

[0072] 3. Preparation of cell wall-breaking enzyme solution Before transferring the fermentation broth to the fermentation tank, use the fermentation tank cooling system to lower the temperature to 10-15℃. Pass the fermentation broth through a ceramic membrane and wash it twice with tap water to ensure as much of the broth is washed away, concentrating it to a cell count of 200 g / L. The membrane-passing process generates heat, so the temperature needs to be lowered to below 20℃. The cooled bacterial suspension is then homogenized using a high-pressure homogenizer at 60-80 kg / m². The homogenization process will raise the temperature, which must not exceed 30℃. The homogenization cycle is repeated twice. Throughout the homogenization process, the pH of the enzyme solution must be controlled, adjusting it to approximately 7.0-7.2 using a sodium hydroxide solution. After homogenization, cool the solution to 0℃, dispense the enzyme solution, and freeze it in a cold storage for later use.

[0073] To construct a 1 L catalytic reaction system, add 176 g of L-AA, 513 g of sucrose, and 25 mL of enzyme solution (equivalent to a wet cell concentration of 50 g / L). Then, add purified water to make up the volume to 1 L. Set the pH to 5.4 and the temperature to 35 ℃.

[0074] The reaction solutions were collected at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, and the strain was subjected to treatment using the method described in Example 4. E. coliBL21(DE3) / pET28a-SPase and strain E. coli The catalytic results of BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C were analyzed by liquid chromatography. The catalytic effect of AA-2G during the reaction process is shown in Table 12. (Strain) E. coli The sucrose phosphorylase prepared by BL21(DE3) / pET28a-SPase was reacted with the enzyme solution for 12 h, resulting in an AA-2G conversion rate of 29.3%, a product concentration of 316.2 g / L, and an impurity content of 0.81%. E. coli The sucrose phosphorylase solution prepared by BL21(DE3) / pET28a-SPase-I189L / P434S / P480S-T4C catalyzed the reaction for 24 h, achieving an AA-2G conversion rate of 93.4% and a product concentration of 316.2 g / L. The AA-2G conversion rate and yield increased threefold, while the impurity content decreased to 0 g / L. The product conversion rate change with reaction time is shown in the figure below. Figure 11 As shown. Immediately after the reaction, the reaction solution was adjusted to pH 3.0-4.0 (citric acid buffer system), and heat-treated at 60℃ for 10 minutes to inactivate the enzyme and precipitate the protein. Centrifugation (8000×g, 15 min) was performed to remove enzyme residue and flocculents. The supernatant was decolorized by adsorbing pigments and hydrophobic impurities through an activated carbon column. Then, nanofiltration was used for concentration, anion exchange resin was used to remove acidic substances, and a gradient elution with macroporous adsorption resin was used to purify AA-2G. Finally, drying and crystallization were performed to obtain AA-2G crystalline powder. The purified AA-2G sample was analyzed by liquid chromatography using the method in Example 4, and the results are shown below. Figure 10 As shown, at this point, only AA-2G is a single peak in the chromatogram, and no impurities are generated at 10.699 min.

[0075] Table 12 Comparison of catalytic reaction effects between the original strain and the mutant strain

[0076] Example 10: Expression of sucrose phosphorylase mutant in Bacillus subtilis Escherichia coli containing the pP43NMK empty vector plasmid was streaked to activate it. Single colonies from the plate were picked and inoculated into liquid LB medium containing 100 µg / mL ampicillin (Diamond, USP Grade). The culture was incubated at 37°C for 12 h to obtain E. coli culture for plasmid extraction. The extraction process followed the instructions of the Qingke Plasmid Mini-Extraction Kit. Linearized vectors were obtained by inverse PCR amplification. Primers pP43NMK-F: 5'-GCAGCACCGCAGGTGCGCA-3' (SEQ ID NO.151) and pP43NMK-R: 5'-AGCTGAGGCATGTGTTACAAAAACCATTTGG-3' (SEQ ID NO.152) were designed and synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0077] Using the extracted pP43NMK empty vector plasmid as a template, PCR was performed using Qingke high-fidelity enzyme and its supporting materials to obtain the linearized fragment of the pP43NMK vector. The PCR system consisted of 50 μL units, and the amounts of each component added were shown in Table 3 of Example 5. The operation was performed under ice bath conditions. After the system was added, it was thoroughly mixed with a small shaker and placed into a PCR instrument for amplification. The PCR reaction process was as follows: pre-denaturation at 95℃ for 5 min; then, a cycle of denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, and holding at 72℃ for 1 min was performed, and this cycle was repeated 30 times; finally, the system was held at 72℃ for 5 min. After the reaction, the PCR product was recovered using a DNA gel purification kit, and the concentration of the recovered PCR product was determined. The product was stored at -20℃.

[0078] Using the sucrose phosphorylase mutant plasmid pET28a-SPase-I189L / P434S / P480S-T4C as a template, primers F1 and F2 carrying homologous arms of the pP43NMK vector were designed. The gene fragment of the sucrose phosphorylase mutant SPase-I189L / P434S / P480S-T4C was amplified using PCR technology. The PCR product was recovered using a DNA gel purification kit, and the concentration of the recovered PCR product was determined. The primers are shown below: F1: 5'-GTTTTTGTAACACATGCCTCAGCTATGAAGAACAAAGTACAGCTGATCAC-3' (SEQ IDNO.146); F2: 5'-CGCACCTGCGGTGCTGCTCAATCGATGTCAGCGATCG-3' (SEQ ID NO. 147); Using a one-step cloning kit, the gene fragment of the obtained sucrose phosphorylase mutant SPase-I189L / P434S / P480S-T4C was ligated to the linearized pP43NMK vector to obtain the recombinant plasmid pP43NMK-SPase-I189L / P434S / P480S-T4C. The ligation volume for one-step cloning was 10 μL, as shown in Table 13. The reaction was carried out at 50 °C for 15 min, and after the reaction, the temperature was lowered to 4 °C or immediately placed on ice for cooling. The recombinant product was stored at -20 °C.

[0079] Table 13 Connection System

[0080] The recombinant plasmid obtained by one-step cloning was transformed into E. coli. E. coli DH5α: Take 10 μL of recombinant plasmid and add it to 50 μL E. coli In DH5α competent cells, gently tap the tube wall to mix, and place on ice for 30 min. Heat shock in a 42℃ water bath for 45 s, then immediately place on ice for 2 min. Add 1 mL of LB liquid medium to the tube and incubate at 37℃ on a shaker for 1 h. Centrifuge the culture at 4500 rpm for 4 min, and collect 800 μL of supernatant. Resuspend the cells in the remaining medium, and spread 100 μL onto LB solid medium containing 100 μg / mL Amp resistance. Incubate overnight at 37℃ for 12–14 h to obtain recombinant *E. coli*. E. coli DH5α-pP43NMK-SPase-I189L / P434S / P480S-T4C. Single colonies of recombinant *E. coli* were streaked onto fresh LB agar containing ampicillin resistance. Single colonies from the streaked plates were then inoculated into liquid LB agar containing 100 µg / mL ampicillin and incubated at 37°C for 12 h. The resulting *E. coli* culture was used for plasmid extraction, yielding the amplified recombinant plasmid pP43NMK-SPase-I189L / P434S / P480S-T4C.

[0081] Recombinant plasmid transformed into Bacillus subtilis B. subtilis WB 600: (1) Required reagents Hypertonic liquid medium: LB liquid medium supplemented with 91 g / L sorbitol (Maclean, reagent grade).

[0082] Electroporation medium: 91 g / L sorbitol, 91 g / L mannitol (Maclean, reagent grade) and pure water.

[0083] RM medium: LB liquid medium supplemented with 91 g / L sorbitol and 69 g / L mannitol.

[0084] (2) Preparation of competent states Bacillus subtilis B. subtilis WB 600 single colonies were cultured overnight at 37°C and 200 rpm in LB medium for 8-10 hours.

[0085] Take 2.5 mL of seed culture and inoculate it into 40 mL of hypertonic liquid culture medium. Incubate at 37℃ and 200 rpm for 4-4.5 h.

[0086] Collect bacterial cells by centrifugation: After incubating on ice for 10 min, centrifuge at 3500 rpm for 5 min at 4℃.

[0087] Washing: Slowly pour in pre-cooled electroporation medium, gently aspirate to suspend, centrifuge and discard the supernatant. Repeat this step 3-4 times.

[0088] Aliquoting: Gently suspend the bacterial cells in 1 mL of electroporation medium, aliquot 100 μL into each tube, and store at -80℃.

[0089] (3) Transformation Set the voltage to 2.4 kV and prepare the electroporation cuvette on ice. Add 1-5 μL of plasmid to Bacillus subtilis competent cells, incubate on ice for 20 min, then transfer to the electroporation cuvette and electroporate at 2.4 kV. Immediately after electroporation, remove the cuvette and add 800 μL of RM medium. Incubate at 37°C and 200 rpm for 3.5 h. Then, spread the revived cells on LB resistant plates and incubate upside down for 12-16 h.

[0090] Recombinant Bacillus subtilis B. subtilis Single colonies of WB 600 / pP43NMK-SPase-I189L / P434S / P480S-T4C were picked and inoculated into NA medium. After incubation at 30°C for 24 h, the bacterial culture was centrifuged, the precipitate was resuspended in water and then disrupted by an ultrasonic homogenizer. The supernatant and the precipitate were used for SDS-PAGE analysis.

[0091] The supernatant and precipitate fragment of recombinant Bacillus subtilis were subjected to catalytic reaction using the method of Example 3. The catalytic results of the supernatant and precipitate fragment of recombinant Bacillus subtilis were detected by liquid phase analysis using the analytical method of Example 4. The final results showed that the catalytic reaction conversion rate of the supernatant was 4.7%, the product yield was 16.1 g / L, and the impurity content was 0 g / L; the catalytic reaction conversion rate of the precipitate fragment was 6.9%, the product yield was 23.3 g / L, and the impurity content was 0 g / L.

[0092] Example 11: Expression of sucrose phosphorylase mutant in Corynebacterium glutamicum Escherichia coli containing the pXMJ19 empty vector plasmid was streaked to activate the culture. Single colonies from the plate were picked and inoculated into liquid LB medium containing 100 µg / mL chloramphenicol (Biofrox) and incubated at 37°C for 12 h to obtain E. coli culture for plasmid extraction. The extraction process followed the instructions of the Qingke Plasmid Mini-Extraction Kit. Linearized vectors were obtained by inverse PCR amplification. Primers pXMJ19-F: 5'-GCAGCACCGCAGGTGCGCA-3' (SEQ ID NO.153) and pXMJ19-R: 5'-AGCTGAGGCATGTGTTACAAAAACCATTTGG-3' (SEQ ID NO.154) were designed and synthesized by Hangzhou Qingke Biotechnology Co., Ltd.

[0093] Using the sucrose phosphorylase mutant plasmid pET28a-SPase-I189L / P434S / P480S-T4C as a template, primers F1 and F2 carrying the homologous arm of the pXMJ19 vector were designed. The gene fragment of the sucrose phosphorylase mutant SPase-I189L / P434S / P480S-T4C was amplified using PCR technology. The PCR product was recovered using a DNA gel purification kit, and the concentration of the recovered PCR product was determined. The primers are shown below: F1: 5'-GTTTTTGTAACACATGCCTCAGCTATGAAGAACAAAGTACAGCTGATCAC-3' (SEQ IDNO.148); F2: 5'-CGCACCTGCGGTGCTGCTCAATCGATGTCAGCGATCG-3' (SEQ ID NO. 149); Using a one-step cloning kit, the gene fragment of the obtained sucrose phosphorylase mutant SPase-I189L / P434S / P480S-T4C was ligated to the pXMJ19 linearized vector to obtain the recombinant plasmid pXMJ19-SPase-I189L / P434S / P480S-T4C. The ligation volume for one-step cloning was 10 μL, as shown in Table 13 of Example 10. The reaction was carried out at 50 °C for 15 min, and after the reaction, the temperature was lowered to 4 °C or immediately placed on ice for cooling. The recombinant product was stored at -20 °C.

[0094] The recombinant plasmid obtained by one-step cloning was transformed into E. coli. E. coli DH5α: Take 10 μL of recombinant plasmid and add it to 50 μL E. coliIn DH5α competent cells, gently tap the tube wall to mix, and place on ice for 30 min. Heat shock in a 42℃ water bath for 45 s, then immediately place on ice for 2 min. Add 1 mL of LB liquid medium to the tube and incubate at 37℃ on a shaker for 1 h. Centrifuge the culture at 4500 rpm for 4 min, and collect 800 μL of supernatant. Resuspend the cells in the remaining medium, and spread 100 μL onto LB solid medium containing 100 μg / mL chloramphenicol resistance. Incubate overnight at 37℃ for 12–14 h to obtain recombinant *E. coli*. E. coli DH5α- pXMJ19-SPase-I189L / P434S / P480S-T4C. Single colonies of recombinant *E. coli* were streaked onto fresh LB agar containing Chl-resistant bacteria. Single colonies from the streaked plates were then inoculated into liquid LB agar containing 100 µg / mL chloramphenicol and incubated at 37°C for 12 h. The resulting *E. coli* culture was used for plasmid extraction, yielding the amplified recombinant plasmid pXMJ19-SPase-I189L / P434S / P480S-T4C.

[0095] Transformation of recombinant plasmid into Corynebacterium glutamicum ATCC: (1) Competent culture medium epo: BHI 38.5 g / L (Haibo Biotechnology), glycine 25 g / L (Maclean, reagent grade), sorbitol 91 g / L, Tween 80 1 g / L (Maclean, reagent grade).

[0096] (2) Preparation of competent Corynebacterium glutamicum cells Single colonies of *Corynebacterium glutamicum* (ATCC) were collected and cultured in BHI medium at 30°C and 200 rpm for 24 h. Then, 1 mL of the colony was transferred to 50 mL of OPO medium and cultured at 30°C and 200 rpm for 3-4 h until OD500 was reached. 600 The concentration was set to 0.8-0.9. After pre-cooling on ice for 30 min, the supernatant was removed by centrifugation. The cells were washed three times with 10% glycerol, and finally, 1 mL of 10% glycerol was added to suspend the bacterial cells. The cells were then aliquoted and stored at -80°C.

[0097] (3) Transformation Pre-cool the electroshock cup before use. Add 800-1000 ng of recombinant plasmid pXMJ19-SPase-I189L / P434S / P480S-T4C to competent cells, incubate on ice for 15-20 min, and electrolyze twice at 1.8 kV. Immediately after electrolysis, remove the electrolysis cuvette and add 1 mL of BHI and sorbitol medium. Incubate at 30℃ and 200 rpm for 3.5 h. Then, spread the revived cells on BHI-resistant plates and incubate upside down for 12-16 h to obtain recombinant Corynebacterium glutamicum ATCC / pXMJ19-SPase-I189L / P434S / P480S-T4C.

[0098] Single colonies of recombinant Corynebacterium glutamicum were picked and inoculated into BHI medium and cultured at 30°C for 24 h. Then, at a volume concentration of 2%, the colonies were transferred to 150 mL of BHI liquid medium containing 100 μg / mL chloramphenicol and cultured at 30°C and 200 rpm until the bacterial concentration reached OD500. 600 To obtain an induction culture medium, add IPTG to a final concentration of 0.8 mM to a final concentration of 0.6-0.8 mM, and incubate at 24°C for 12 h. Centrifuge the obtained culture medium, resuspend the precipitate in water, and then disrupt it using an ultrasonic homogenizer. The disrupted precipitate is used for SDS-PAGE analysis.

[0099] The catalytic reaction of the precipitate and broken liquid of recombinant Corynebacterium glutamicum was carried out using the method of Example 3. The catalytic results of the precipitate and broken liquid of recombinant Corynebacterium glutamicum were detected by liquid phase analysis using the analytical method of Example 4. Finally, the conversion rate of the catalytic reaction of the precipitate and broken liquid was 0.5%, the product concentration was 1.6 g / L, and the impurity content was 0 g / L.

[0100] By constructing recombinant genetically engineered bacteria using various host bacteria to express sucrose phosphorylase mutants and catalyze the conversion of L-AA to AA-2G, the results showed that recombinant genetically engineered bacteria constructed using Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum as hosts could all express sucrose phosphorylase mutants and catalyze the conversion of L-AA to AA-2G. However, the catalytic effects of the engineered bacteria constructed using different hosts varied. Among them, the recombinant genetically engineered bacteria constructed using Escherichia coli as the host showed the best catalytic performance for the preparation of AA-2G.

[0101] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A sucrose phosphorylase mutant, characterized in that, The mutant is based on the sequence of sucrose phosphorylase as shown in SEQ ID No. 3 and has a mutation site, which includes any one or more of A272V, P434S, E63D, I189L, P480S, T288S, D168A, D447E, T455S or G459S.

2. The mutant as described in claim 1, characterized in that, The mutant was obtained by mutating three sites, P434S, I189L and P480S, in the amino acid sequence shown in SEQ ID No.

3.

3. The mutant as described in claim 2, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.

4.

4. A recombinant gene expression vector, characterized in that, The nucleotide sequence of the mutant as described in any one of claims 1 to 3.

5. The recombinant gene expression vector as described in claim 4, characterized in that, The promoter of the recombinant gene expression vector is a T7 promoter mutant, which is obtained by any one or more mutations of T1A, A2T, T4A, T4C or G7A in the sequence shown in the nucleotide sequence TAATACGACTCACTATAG.

6. The recombinant gene expression vector as described in claim 5, characterized in that, The T7 promoter mutant was obtained by mutating T4C in the nucleotide sequence TAATACGACTCACTATAG.

7. A recombinant genetically engineered bacterium, characterized in that, The recombinant genetically engineered bacteria comprises the recombinant gene expression vector of any one of claims 4 to 6.

8. The recombinant genetically engineered bacteria as described in claim 7, characterized in that, The host bacteria of the recombinant genetically engineered bacteria are one or more of Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum.

9. The use of the sucrose phosphorylase mutant according to any one of claims 1 to 3, the recombinant gene expression vector according to any one of claims 4 to 6, or the recombinant genetically engineered bacteria according to any one of claims 7 to 8 in the preparation of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

10. The use of the sucrose phosphorylase mutant according to any one of claims 1 to 3, the recombinant gene expression vector according to any one of claims 4 to 6, or the recombinant genetically engineered bacteria according to any one of claims 7 to 8 in the preparation of a catalyst that improves the conversion rate and yield of 2-O-α-D-glucopyranosyl-L-ascorbic acid and reduces the impurity content.

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