Recombinant escherichia coli for producing sucrose phosphorylase as well as construction method and application of recombinant escherichia coli

Abstract

The invention discloses recombinant escherichia coli for producing sucrose phosphorylase as well as a construction method and application of the recombinant escherichia coli, and belongs to the technical field of biological engineering. The sucrose phosphorylase is constructed, the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO.11, a recombinant escherichia coli BdSP-L341V / L343F / V346P for expressing the sucrose phosphorylase is constructed, the sucrose phosphorylase expressed by recombinant bacteria or whole cells of the recombinant bacteria are used for carrying out catalytic reaction, and efficient production of ascorbyl glucoside can be realized. The invention also provides a method for producing ascorbic acid glucoside, the catalytic reaction is carried out at normal temperature and normal pressure, toxic and harmful organic solvents are not used, and the pollution to the environment is reduced; the whole catalytic process is a one-step reaction, has the advantages of high conversion rate, simple purification step and simple process, can better save the production cost, and has wide industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to a recombinant Escherichia coli producing sucrose phosphorylase, a construction method and an application thereof. Background Art

[0002] Ascorbic acid (L-AA), also known as vitamin C (VC), is widely found in vegetables, fresh fruits, supplements, and green parts of plants. As an essential nutrient for human health, L-AA possesses antioxidant, whitening, anti-inflammatory, moisturizing, and immune-boosting properties. However, its application is greatly limited due to its extreme instability in the presence of air, light, heat, and metal ions, and its excessive absorption can lead to cytotoxicity. Ascorbyl glucoside (AA-2G) has begun to gain attention due to its superior physical and chemical properties.

[0003] As a recognized whitening additive, AA-2G boasts a more pronounced and effective whitening effect than other similar products. It addresses daily skin whitening needs by directly removing melanin from the epidermis through reverse melanin reduction. Therefore, it is often used as a key ingredient in skincare products for whitening, freckle removal, antioxidants, and anti-aging. To prolong collagen synthesis and maintain skin elasticity and health, it has become a mainstream ingredient in the whitening market. In healthcare, AA-2G can lower cholesterol levels, prevent viral and bacterial infections, enhance immunity, fight cancer, and prevent scurvy. It is also a key ingredient in anti-allergy and urinary tract infection medications.

[0004] Early researchers focused on chemical synthesis of AA-2G, which is time-saving and fast. However, due to its cumbersome and unsustainable steps, it has been gradually replaced by enzymatic synthesis in recent years. Compared with traditional chemical synthesis, enzymatic synthesis of AA-2G offers advantages such as mild reaction conditions and readily available and inexpensive substrates. Enzymatic catalysis also offers greater specificity and higher yields. Sucrose phosphorylase (SPase) is a commonly used enzyme in the enzymatic synthesis of AA-2G. Transglycosylation of the substrate by SPase significantly improves its water solubility, antioxidant properties, and biological activity. Therefore, in-depth research on the biosynthesis of AA-2G is crucial to improve synthesis efficiency and reduce production costs, which is of great significance for the industrial development of AA-2G biosynthesis.

[0005] The invention patent with application number 201910754470.X discloses an Escherichia coli that produces sucrose phosphorylase. The invention uses pET-28a as a vector to construct a recombinant plasmid pET-28aSPase with the nucleotide sequence encoding the gene, which is transformed into Escherichia coli BL21 (DE3) to construct a recombinant Escherichia coli, ferment to produce recombinant sucrose phosphorylase, and mutate the constructed recombinant bacteria by ultraviolet mutagenesis to screen out an Escherichia coli strain with high sucrose phosphorylase production. The enzyme activity of the fermentation intracellular cell wall-broken supernatant is 819.16U / mL, and the specific enzyme activity is 206.91U / mg. However, the mutation caused by ultraviolet mutagenesis is reversible, and there is a hidden danger of photorepair, which is not conducive to the long-term use of the strain. In addition, the enzyme activity produced is unstable and cannot be used for the production and conversion of sucrose phosphorylase. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant Escherichia coli producing sucrose phosphorylase and its construction method and application to solve the problems existing in the above-mentioned prior art. The sucrose phosphorylase expressed by the recombinant Escherichia coli of the present invention can efficiently produce ascorbyl glucoside.

[0007] The present invention discloses a sucrose phosphorylase. The amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO.11.

[0008] The present invention also discloses a gene encoding the sucrose phosphorylase. The nucleotide sequence of the gene encoding the sucrose phosphorylase is shown as SEQ ID NO.10.

[0009] The invention also discloses the application of the sucrose phosphorylase or the encoding gene in constructing a recombinant Escherichia coli producing sucrose phosphorylase.

[0010] The present invention also discloses a method for constructing the above-mentioned recombinant Escherichia coli BdSP-L341V / L343F / V346P that produces sucrose phosphorylase, comprising the following steps:

[0011] (1) First, the coding gene is connected to the expression vector to construct a recombinant plasmid. The nucleotide sequence of the coding gene is shown in SEQ ID NO. 9, and the amino acid sequence encoded by the gene is shown in SEQ ID NO. 12;

[0012] (2) The recombinant plasmid obtained in step (1) is transformed into competent Escherichia coli, the plasmid is extracted and used as a template to mutate the codon CTG encoding leucine at position 341 in SEQ ID NO.9 to the codon GTG encoding valine, the codon CTG encoding leucine at position 343 to the codon TTC encoding phenylalanine, and the codon GTG encoding valine at position 346 to the codon CCG encoding proline, thereby obtaining the BdSP-L341V / L343F / V346P gene with the mutated nucleotide sequence of SEQ ID NO.10; and then, after sequencing verification and screening, the recombinant Escherichia coli BdSP-L341V / L343F / V346P is obtained.

[0013] Preferably, the expression vector in step (1) is pET-28a(+).

[0014] In any of the above schemes, preferably, the competent Escherichia coli in step (2) is a competent cell BL21 (DE3).

[0015] The present invention also discloses a recombinant Escherichia coli BdSP-L341V / L343F / V346P, which is constructed by using any one of the construction methods described above.

[0016] The invention also discloses the use of sucrose phosphorylase, encoding gene or recombinant Escherichia coli BdSP-L341V / L343F / V346P in catalytic synthesis of ascorbyl glucoside.

[0017] The present invention also discloses an enzymatic method for producing ascorbyl glucoside, wherein sucrose and ascorbic acid are used as substrates and sucrose phosphorylase is used to carry out a catalytic reaction to obtain the ascorbyl glucoside. The amino acid sequence of the sucrose phosphorylase is shown in SEQ ID NO.11.

[0018] The present invention also discloses a whole-cell method for producing ascorbyl glucoside, wherein sucrose and ascorbic acid are used as substrates, and whole cells of the recombinant Escherichia coli BdSP-L341V / L343F / V346P obtained by any of the construction methods described above are used to carry out a catalytic reaction to obtain the ascorbyl glucoside.

[0019] The present invention discloses a recombinant Escherichia coli that produces sucrose phosphorylase, a construction method, and an application thereof, belonging to the field of bioengineering technology. The present invention constructs a sucrose phosphorylase, the amino acid sequence of which is shown in SEQ ID NO.11, and simultaneously constructs a recombinant Escherichia coli BdSP-L341V / L343F / V346P strain expressing the sucrose phosphorylase. The sucrose phosphorylase expressed by the recombinant bacteria or the whole cells of the recombinant bacteria are used to carry out a catalytic reaction to achieve efficient production of ascorbyl glucoside. The sucrose phosphorylase expressed by the recombinant bacteria can produce ascorbyl glucoside with a conversion rate of up to 91.48%; the whole cells of the recombinant bacteria can produce ascorbyl glucoside with a conversion rate of up to 62.20%. The present invention also provides a method for producing ascorbyl glucoside. The catalytic reaction is carried out at room temperature and pressure, and no toxic or harmful organic solvents are used, thereby reducing pollution to the environment. The entire catalytic process is a one-step reaction, which has the advantages of high conversion rate, concise purification steps and simple process, can better save production costs, and has broad industrial application prospects.

[0020] The present invention discloses a method for producing ascorbyl glucoside by using an enzymatic method or a whole-cell method. The entire catalytic reaction is carried out at room temperature and pressure, which is environmentally friendly. No toxic or harmful organic solvents are used, thereby reducing environmental pollution. The entire catalytic process is a one-step reaction, which has the advantages of high conversion rate, concise purification steps and simple procedures, thereby better saving production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the process for constructing recombinant Escherichia coli BdSP-L341V / L343F / V346P in Example 1;

[0022] Figure 2 This is a diagram of the process of synthesizing ascorbyl glucoside catalyzed by sucrose phosphorylase produced by recombinant Escherichia coli BdSP-L341V / L343F / V346P in Examples 4 and 5;

[0023] Figure 3 This is the liquid chromatogram of the standard product of ascorbyl glucoside in Example 6;

[0024] Figure 4 This is the liquid chromatography peak diagram of the reaction of sucrose and ascorbic acid catalyzed by the recombinant Escherichia coli BdSP-L341V / L343F / V346P in Example 6. DETAILED DESCRIPTION

[0025] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0027] In the following examples or comparative examples, Escherichia coli DH5α and Escherichia coli BL21 (DE3) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; and the vector pET-28a-(+) was purchased from Novagen.

[0028] The culture medium formula used is as follows:

[0029] The components and concentrations in LB medium were as follows: yeast extract 5 g / L, tryptone 10 g / L, and sodium chloride 10 g / L.

[0030] The components and concentrations of TB medium were as follows: tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 mL, potassium dihydrogen phosphate trihydrate 16.43 g / L, potassium dihydrogen phosphate 2.31 g / L;

[0031] DNS Reagent Recipe: Weigh 20.05 g of solid NaOH and dissolve it in a volumetric flask to 200 mL to obtain a 10% NaOH solution. Weigh 4.41 g of solid DNS and dissolve it in the solution. Dilute the solution to 440 mL to obtain a 1% DNS solution. Solution A: Dissolve 6.9 g of crystalline phenol in 15.2 mL of 10% NaOH solution, then dilute the solution to 69 mL with water. Add 6.9 g of NaHSO₃ to this solution. Solution B: Weigh 255 g of potassium sodium tartrate and add it to 300 mL of 10% NaOH solution. Add 880 mL of 1% 3,5-dinitrosalicylic acid solution. Mix the two solutions to obtain a yellow reagent. Store in a brown bottle and store at room temperature for 7-10 days before use.

[0032] Determination of sucrose phosphorylase activity:

[0033] The hydrolytic activity assay involves adding 1.8 mL of a mixed solution (5% (w / v) sucrose, 20 mmol / L sodium citrate buffer, pH 6.0) and 0.2 mL of enzyme solution to a 55°C water bath for 30 minutes. The reaction is then terminated by boiling for 10 minutes. 250 μL of the reaction solution and 750 μL of DNS are then boiled for 5 minutes. 5 mL of pure water is then immediately added and cooled. The absorbance at 520 nm is measured using a UV-visible spectrophotometer, and the amount of fructose is calculated based on the absorbance. One unit (U) of SPase hydrolytic activity is defined as the amount of enzyme required to generate 1 μmol of fructose per minute.

[0034] Enzyme activity calculation formula: Enzyme activity (U / mL) = (OD 520 × dilution factor) / 0.04267

[0035] The transglycosylation activity assay involves adding 4 mL of a mixed solution (100 mmol / L ascorbic acid, 800 mmol / L sucrose, 20 mmol / L sodium citrate buffer, pH 4.6) to 1 mL of enzyme solution. The reaction is incubated at 45°C for 1 hour, then boiled for 10 minutes to terminate the reaction. The amount of AA-2G produced is then calculated using high-performance liquid chromatography. One unit (U) of SPase transglycosylation activity is defined as the amount of enzyme required to generate 1 μmol of AA-2G per minute.

[0036] Example 1

[0037] A variety of sucrose phosphorylase genes were searched from NCBI. After comparison, a gene (BAQ26093.1) from Bifidobacterium dentium JCM 1195 was obtained. After codon optimization, the gene BdSP was synthesized. According to the sequences of the BdSP gene and the pET-28a-(+) plasmid, primers were designed using Primer Premier 5.0. The forward primer (5'-3') sequence was SEQ ID NO.1, and the reverse primer sequence was SEQ ID NO.2. PCR amplification was performed to obtain a truncated gene clone fragment BdSP (SEQ ID NO.9) containing BamH I and Xho I restriction sites. The above gene clone fragment BdSP was digested with double enzymes (BamH I and Xho I). I restriction enzyme cutting site) was ligated into the pET-28a-(+) plasmid to obtain the recombinant plasmid pET-28a-(+)-BdSP, and the recombinant plasmid was introduced into BL21(DE3) competent cells. The plasmid was extracted and used as a template to firstly perform a mutation at amino acid position 341 using a forward primer (primer sequence is SEQ ID NO.3) and a reverse primer (primer sequence is SEQ ID NO.4) to mutate the codon encoding leucine (CTG) to a codon encoding valine (GTG). Then, a mutation at amino acid position 343 was performed using a forward primer (primer sequence is SEQ ID NO.5) and a reverse primer (primer sequence is SEQ ID NO.6) to mutate the codon encoding leucine (CTG) to a codon encoding phenylalanine (TTC). Then, a mutation was performed using a forward primer (primer sequence is SEQ ID NO.7) and a reverse primer (primer sequence is SEQ ID NO.8). ID NO.8) to mutate the amino acid at position 346, so that the codon encoding valine (GTG) was mutated to the codon encoding proline (CCG) to obtain the mutated gene BdSP-L341V / L343F / V346P (primer sequence is SEQ ID NO.10), which was introduced into a DMT competent cell to extract the plasmid and sequenced for verification. After verification, the plasmid was introduced into a BL21 (DE3) competent cell and screened on a kanamycin-resistant plate to obtain the recombinant Escherichia coli BdSP-L341V / L343F / V346P that produces sucrose phosphorylase. The schematic diagram of the process for constructing the recombinant Escherichia coli BdSP-L341V / L343F / V346P is shown below. Figure 1 shown.

[0038] The nucleotide sequence of BdSP is shown in SEQ ID NO. 9. The sequence corresponding to the mutation is CTG GAT CTG TATCAA GTG, The codon encoding leucine (CTG) and the codon encoding valine (GTG).

[0039] The nucleotide sequence of BdSP-L341V / L343F / V346P is shown in SEQ ID NO.10: The sequence corresponding to the mutation is GTG GAT TTC TATCAA CCG The two original codons encoding leucine (CTG) mutated into codons encoding valine (GTG) and phenylalanine (TTC), and the original codon encoding valine (GTG) mutated into a codon encoding proline (CCG).

[0040] Table 1 Primer sequences involved in Example 1

[0041]

[0042] Example 2

[0043] The recombinant E. coli BdSP-L341V / L343F / V346P from Example 1 was inoculated into 50 mL LB medium containing 100 μg / mL kanamycin sulfate and cultured at 37°C, 220 rpm for 12 h until the absorbance reached OD 600 4.0 to obtain seed solution; take 1 mL of seed solution to 50 mL of TB medium and culture at 37 ° C, 220 rpm for 4-6 h until the absorbance OD 600 The pH value was 4.0, and IPTG inducer at a concentration of 62.5 μg / mL was added. The fermentation was induced at 25 °C for 16 h, and the culture was centrifuged at 8000 rpm at 4 °C for 15 min. The supernatant was removed, and MES buffer with a pH of 6.0 was added to prepare a 0.1 g / mL whole-cell suspension with a hydrolase activity of 400 U / mL.

[0044] Example 3

[0045] The recombinant E. coli BdSP-L341V / L343F / V346P from Example 1 was inoculated into 50 mL LB medium containing 100 μg / mL kanamycin sulfate and cultured at 37°C, 220 rpm for 12 h until the absorbance reached OD 600 4.0 to obtain seed solution; take 1 mL of seed solution to 50 mL of TB medium and culture at 37°C, 220 rpm for 4-6 h until the absorbance OD 600The cell suspension was stirred at a pH of 4.0, and IPTG was added at a concentration of 62.5 μg / mL. Fermentation was induced at 25°C for 16 hours. The cells were centrifuged at 8000 rpm for 15 minutes at 4°C. The supernatant was removed, and MES buffer (pH 6.0) was added to prepare a 0.1 g / mL whole-cell suspension with a hydrolase activity of 400 U / mL. The suspension was then ultrasonicated at 175W for 15 minutes in an ice-water bath. The supernatant was separated by centrifugation to obtain the crude enzyme solution with a hydrolase activity of 1000 U / mL. The amino acid sequence of sucrose phosphorylase expressed by recombinant Escherichia coli BdSP-L341V / L343F / V346P is shown in SEQ ID NO. 11.

[0046] Example 4

[0047] A 100 mmol / L L-AA and 800 mmol / L sucrose mixed solution was prepared using a citric acid-sodium citrate buffer solution at pH 4.6. The pH of the substrate solution was adjusted to 4.6 using a 20% sodium carbonate solution. The whole cell suspension prepared in Example 2 was added to the reaction system to obtain an enzyme activity of 30 U / mL. The mixture was stirred at 45°C for 12 h to catalyze the synthesis of ascorbyl glucoside. The catalytic synthesis process is as follows: Figure 2 As shown, the conversion rate is 62.20%.

[0048] Example 5

[0049] A 100 mmol / L L-AA and 800 mmol / L sucrose mixed solution was prepared using a citric acid-sodium citrate buffer solution at pH 4.6. The pH of the substrate solution was adjusted to 4.6 using a 20% sodium carbonate solution. The crude enzyme solution prepared in Example 3 was added to the reaction system to obtain an enzyme activity of 30 U / mL. The mixture was stirred at 45°C for 12 h to catalyze the synthesis of ascorbyl glucoside. The catalytic synthesis process is as follows: Figure 2 As shown, the conversion rate was 91.48%.

[0050] Example 6

[0051] A mixed solution of 100 mmol / L L-AA and 800 mmol / L sucrose was prepared using citric acid-sodium citrate buffer with a pH of 4.6. The pH of the substrate solution was adjusted to 4.6 with 20% sodium carbonate solution. The crude enzyme solution prepared in Example 3 was added, and the pH of the reaction solution was maintained within 4.6±0.05. When the pH of the reaction solution no longer changed, the reaction was complete. The reaction was terminated by heating at 100°C for 5 min. A certain amount of the reaction solution was centrifuged to remove the precipitate. The reaction solution was diluted with 20 mM potassium dihydrogen phosphate solution, and the supernatant was filtered using a 0.22 μm filter membrane. The filtered supernatant was detected by high performance liquid chromatography (detection conditions: chromatographic column: Alphasil VC-C18 column (4.6 mm×25 cm, 5 μm); column temperature: 25°C, detection wavelength: 240 nm, flow rate: 0.5 mL / min; injection volume: 20 μL; mobile phase: 20 mM KH2PO4 (using phosphoric acid to adjust pH to 2.0). The liquid chromatography peak diagram of the reaction solution is as follows Figure 4 As shown; 0.169 g / mL ascorbyl glucoside standard solution was prepared using 20 mM potassium dihydrogen phosphate solution, and the same high performance liquid chromatography was used to detect the ascorbyl glucoside standard solution. The liquid chromatogram of the ascorbyl glucoside standard solution is shown in FIG. Figure 3 shown.

[0052] Comparative Example 1

[0053] Similar to Example 5, except that the crude enzyme solution prepared in Example 3 was replaced with the crude enzyme solution prepared by the following method:

[0054] The recombinant plasmid pET-28a-(+)-BdSP of Example 1 was introduced into BL21(DE3) competent cells, and the recombinant Escherichia coli BdSP was screened on a kanamycin resistance plate. The recombinant Escherichia coli BdSP was inoculated into 50 mL LB medium containing 100 μg / mL kanamycin sulfate and cultured at 37°C, 220 rpm for 12 h until the absorbance OD value reached 0. 600 4.0 to obtain seed solution; take 1 mL of seed solution to 50 mL of TB medium and culture at 37 ° C, 220 rpm for 4-6 h until the absorbance OD 600 The concentration of IPTG inducer was 4.0, and 62.5 μg / mL IPTG inducer was added. The fermentation was induced at 25 ℃ for 16 h, and the supernatant was removed at 8000 rpm at 4 ℃ for 15 min. The supernatant was removed and MES buffer with pH = 6.0 was added. An ice-water bath was added, and the supernatant was obtained by ultrasonication at 175W for 15 min. The supernatant was obtained by centrifugation, i.e., the crude enzyme solution with a hydrolytic activity of 120 U / mL.

[0055] A mixed solution of 100 mmol / L L-AA and 800 mmol / L sucrose was prepared using citric acid-sodium citrate buffer at pH 4.6. The pH of the substrate solution was adjusted to 4.6 with 20% sodium carbonate solution. The crude enzyme solution prepared in Example 3 was added to obtain an enzyme activity of 30 U / mL in the reaction system. The mixture was stirred at 45°C for 12 h to catalyze the synthesis of ascorbyl glucoside with a conversion rate of 31.27%.

[0056] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A sucrose phosphorylase, characterized in that The amino acid sequence of the sucrose phosphorylase is shown in SEQ ID NO.

11.

2. The gene encoding sucrose phosphorylase according to claim 1, wherein The nucleotide sequence of the coding gene is shown in SEQ ID NO.

10.

3. Use of the sucrose phosphorylase according to claim 1 or the encoding gene according to claim 2 in constructing a recombinant Escherichia coli producing sucrose phosphorylase.

4. A method for constructing the recombinant Escherichia coli BdSP-L341V / L343F / V346P producing sucrose phosphorylase according to claim 1, characterized in that: The following steps are involved: (1) First, the coding gene is connected to the expression vector to construct a recombinant plasmid. The nucleotide sequence of the coding gene is shown in SEQ ID NO. 9, and the amino acid sequence encoded by the gene is shown in SEQ ID NO. 12; (2) The recombinant plasmid obtained in step (1) is transformed into competent Escherichia coli, the plasmid is extracted and used as a template to mutate the codon CTG encoding leucine at position 341 in SEQ ID NO.9 to the codon GTG encoding valine, the codon CTG encoding leucine at position 343 to the codon TTC encoding phenylalanine, and the codon GTG encoding valine at position 346 to the codon CCG encoding proline, thereby obtaining the BdSP-L341V / L343F / V346P gene with the mutated nucleotide sequence of SEQ ID NO.10; and then, after sequencing verification and screening, the recombinant Escherichia coli BdSP-L341V / L343F / V346P is obtained.

5. The method for constructing a recombinant Escherichia coli according to claim 4, wherein: The expression vector in step (1) is pET-28a(+).

6. The method for constructing a recombinant Escherichia coli according to claim 4, wherein: The competent Escherichia coli in step (2) is the competent cell BL21 (DE3).

7. A recombinant Escherichia coli BdSP-L341V / L343F / V346P, constructed by the construction method according to any one of claims 4 to 6.

8. Use of the sucrose phosphorylase according to claim 1, the encoding gene according to claim 2, or the recombinant Escherichia coli BdSP-L341V / L343F / V346P according to claim 7 in catalyzing the synthesis of ascorbyl glucoside.

9. A method for producing ascorbyl glucoside by enzymatic method, characterized in that: Sucrose and ascorbic acid are used as substrates, and sucrose phosphorylase is used to carry out a catalytic reaction to obtain the ascorbyl glucoside. The amino acid sequence of the sucrose phosphorylase is shown in SEQ ID NO.

11.

10. A method for producing ascorbyl glucoside using a whole-cell method, characterized in that: Using sucrose and ascorbic acid as substrates, the whole cells of the recombinant Escherichia coli BdSP-L341V / L343F / V346P obtained by the construction method according to any one of claims 4 to 6 are used to carry out a catalytic reaction to obtain the ascorbyl glucoside.

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