Sucrose phosphorylase mutant and its application in the synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a sucrose phosphorylase mutant and its application. The present invention simultaneously mutates multiple amino acid sites at positions 91, 154, 185, 323, and 424 of the wild-type sucrose phosphorylase derived from Bifidobacterium pseudolongum Bifidobacterium pseudolongum ), thereby obtaining a sucrose phosphorylase mutant, and significantly improving its efficiency in producing 2-O-α-D-glucopyranosyl-L-ascorbic acid using sucrose and vitamin C as raw materials. It only takes 15 h to completely catalyze 1.2 M of vitamin C to produce 2-O-α-D-glucopyranosyl-L-ascorbic acid; the yield of 2-O-α-D-glucopyranosyl-L-ascorbic acid reaches 358 g / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a sucrose phosphorylase mutant and its application in the synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] L-ascorbic acid (L-AA), also known as vitamin C, is an important nutrient with antioxidant and neuroprotective properties. Due to the hydroxyl group at the C2 position of L-ascorbic acid being prone to oxidation reactions, L-ascorbic acid is extremely unstable during storage and use. 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), as a substitute for L-ascorbic acid, exhibits better stability in an environment where oxygen, heat, and metal ions are present compared to L-ascorbic acid. In addition, compared with other L-ascorbic acid derivatives, 2-O-α-D-glucopyranosyl-L-ascorbic acid has excellent stability and solubility. When acting, it is hydrolyzed by enzymes in the skin or in vivo (such as α-glucosidase), slowly releasing vitamin C and continuously exerting its efficacy; it is often used as the main ingredient in skin care products such as whitening, freckle removal, antioxidant, and anti-aging.

[0004] 2-O-α-D-glucopyranosyl-L-ascorbic acid is mainly synthesized by an enzymatic method, using an enzyme to catalyze the reaction between vitamin C and a glycosyl donor, with mild conditions and environmental friendliness; it can also be synthesized chemically, but chemical synthesis has problems such as more impurities, a complex purification process, many impurities, and serious pollution. Therefore, the currently mainly used highly specific enzymatic method is adopted for synthesis. Using sucrose and vitamin C as raw materials, 2-O-α-D-glucopyranosyl-L-ascorbic acid is produced under the action of sucrose phosphorylase. However, the currently reported sucrose phosphorylase and its mutants have problems such as low enzyme activity and insufficient stability. At the same time, there is also a problem of low yield of 2-O-α-D-glucopyranosyl-L-ascorbic acid, so it does not meet the requirements of industrial production. Summary of the Invention

[0005] In order to overcome the above problems, the present invention provides a sucrose phosphorylase mutant and its application in the synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

[0006] In the first aspect of the present invention, there is provided a sucrose phosphorylase mutant, which is obtained by simultaneous multi-site mutagenesis of tryptophan at position 91, valine at position 154, histidine at position 185, alanine at position 323, and asparagine at position 424 of the wild-type sucrose phosphorylase with the amino acid sequence shown in SEQ ID NO. 2.

[0007] In one or more embodiments, the sucrose phosphorylase mutant is such that tryptophan at position 91 of the wild-type sucrose phosphorylase with the amino acid sequence shown in SEQ ID NO. 2 is mutated to alanine, valine at position 154 is mutated to threonine, histidine at position 185 is mutated to glycine, alanine at position 323 is mutated to histidine, and asparagine at position 424 is mutated to leucine.

[0008] In the second aspect of the present invention, there is provided a gene encoding the sucrose phosphorylase mutant described in the first aspect.

[0009] In the third aspect of the present invention, there is provided an expression cassette comprising the gene described in the second aspect.

[0010] In the fourth aspect of the present invention, there is provided a recombinant expression vector comprising the gene described in the second aspect.

[0011] In the fifth aspect of the present invention, there is provided a recombinant bacterium comprising the gene described in the second aspect.

[0012] In the sixth aspect of the present invention, there is provided a transgenic cell line comprising the gene described in the second aspect.

[0013] In the seventh aspect of the present invention, there is provided the use of the sucrose phosphorylase mutant described in the first aspect, or the gene described in the second aspect, or the recombinant bacterium described in the fifth aspect in the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

[0014] In the eighth aspect of the present invention, there is provided a method for synthesizing 2-O-α-D-glucopyranosyl-L-ascorbic acid, comprising:

[0015] Using the wet cells obtained by induced culture of the genetically engineered bacterium of the sucrose phosphorylase mutant, or the crude enzyme solution extracted by ultrasonic disruption of the wet cells, or the immobilized enzyme as a catalyst, using sucrose and vitamin C as substrates, and pure water as a reaction medium to form a reaction system, and reacting to obtain 2-O-α-D-glucopyranosyl-L-ascorbic acid;

[0016] wherein the genetically engineered bacterium is constructed by introducing the sucrose phosphorylase mutant described in the first aspect into a host bacterium.

[0017] In one or more embodiments, the dosage of the catalyst is 5 g / L to 25 g / L based on the total weight of the wet cells, the final concentration of the substrate sucrose is 1.2 M to 1.4 M, preferably 1.3 M; the final concentration of the substrate vitamin C is 1.1 M to 1.3 M, preferably 1.2 M.

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) The present invention provides a sucrose phosphorylase mutant and its application. By simultaneously mutating multiple amino acid sites at positions 91, 154, 185, 323, and 424 of the wild-type sucrose phosphorylase derived from Bifidobacterium pseudolongum ( Bifidobacterium pseudolongum ), a sucrose phosphorylase mutant is obtained, thereby significantly improving the efficiency of producing 2-O-α-D-glucopyranosyl-L-ascorbic acid using sucrose and vitamin C as raw materials. It only takes 15 h to catalyze 1.2 M of vitamin C to produce 2-O-α-D-glucopyranosyl-L-ascorbic acid, shortening the production cycle; the yield of 2-O-α-D-glucopyranosyl-L-ascorbic acid reaches 358 g / L, greatly increasing the product yield.

[0020] (2) The content reduction degree of the sucrose phosphorylase mutant provided by the present invention is significantly lower than that of the original strain at a temperature of 40 °C and a pH of 5.0, showing good stability; therefore, the sucrose phosphorylase mutant provided by the present invention has better application value in the production field of 2-O-α-D-glucopyranosyl-L-ascorbic acid. Description of the Drawings

[0021] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0022] Figure 1 For E.coli the reaction formula of the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid by BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L;

[0023] Figure 2 For E.coli the reaction process diagram of the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid by BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L. Detailed Embodiments

[0024] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0026] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0027] The culture medium formulations used in the following examples are as follows:

[0028] LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, solvent is water, pH is 7.4.

[0029] LB plate: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, 18 g / L agar, solvent is water, pH is 7.4.

[0030] The concentration of the product 2-O-α-D-glucopyranosyl-L-ascorbic acid was detected by high performance liquid chromatography (HPLC), and the analysis method was as follows:

[0031] Chromatographic column model: QS-C18, 5 μm, 4.6×250 mm; mobile phase is 20 mM potassium dihydrogen phosphate solution, adjusted to pH = 2 with phosphoric acid, injection volume 10 μL; ultraviolet detector; detection wavelength: 242 nm; detection time: 10 min; flow rate: 0.8 mL / min; column temperature: 30 °C.

[0032] Sample treatment: Take 5 μL of the sample after the reaction ends, dilute it 600 times with an aqueous solution, filter it through a 0.22 μm filter membrane, and perform HPLC detection.

[0033] Example 1

[0034] Construction of expression vector and engineering bacteria:

[0035] Through the mining of the gene library, a strain derived from Bifidobacterium pseudolongum was screened ( Bifidobacterium pseudolongumSucrose phosphorylase (BpSPase) with the NCBI accession number WP_129853343.1 was entrusted to Nanjing Genscript Biotech Co., Ltd. for full gene synthesis. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO:2.

[0036] Primers F1, R1, F2, and R2 were designed according to the nucleotide sequence shown in SEQ ID NO.1 and the pET-28a vector sequence; the nucleotide sequence of F1 is shown in SEQ ID NO.3, the nucleotide sequence of R1 is shown in SEQ ID NO.4, the nucleotide sequence of F2 is shown in SEQ ID NO.5, and the nucleotide sequence of R2 is shown in SEQ ID NO.6.

[0037] F1: 5’-ctttaagaaggagatataccATGAAAAATAAGGTACAACTAATAACATATGC-3’;

[0038] R1: 5’-tggtggtggtggtgctcgagTTAATCCATGTGTGCTACCGGC-3’;

[0039] F2: 5’-CTCGAGCACCACCACCACC-3’;

[0040] R2: 5’-GGTATATCTCCTTCTTAAAGTTAAACAAAAT-3’;

[0041] Using the pET-28a plasmid as an expression vector, Escherichia coli E.coli BL21(DE3) / pET-28a-BpSPase was constructed.

[0042] Construction of the expression plasmid: Under the priming of primers F1 / R1 and F2 / R2, using the target gene as a template, the sucrose phosphorylase gene sequence with homologous arms was amplified using high-fidelity Pfu DNA polymerase. Using the pET-28a plasmid as a template, the linearized vector sequence was amplified using high-fidelity Pfu DNA polymerase, and the target gene was homologously recombined with the linearized vector using a homologous recombinase to construct the plasmid pET28a-BpSPase.

[0043] Preparation of competent cells: Obtained from a glycerol tube stored in a -80 °C refrigerator E. coliBL21(DE3) strain was streaked on an antibiotic-free LB plate and cultured at 37 °C for 10 h to obtain single colonies; single colonies on the LB plate were picked and inoculated into a test tube containing 5 mL of LB liquid medium, and cultured at 37 °C and 180 rpm for 9 h; 200 μL of the bacterial solution was taken from the test tube and inoculated into 50 mL of LB liquid medium, and cultured at 37 °C and 180 rpm until OD 600 reached 0.4 - 0.6; the bacterial solution was pre-cooled on ice, the bacterial solution was taken into a sterilized centrifuge tube, placed on ice for 10 min, centrifuged at 4 °C and 5000 rpm for 10 min; the supernatant was poured out, taking care to prevent contamination, the precipitated cells were resuspended with pre-cooled 0.1 mol / L CaCl2 aqueous solution and placed on ice for 30 min; centrifuged at 4 °C and 5000 rpm for 10 min, the supernatant was discarded, the precipitated cells were resuspended with pre-cooled 0.1 mol / L CaCl2 aqueous solution containing 15% glycerol, 100 μL of the resuspended cells was aliquoted into sterilized 1.5 mL centrifuge tubes and stored in a -80 °C refrigerator and taken out when needed.

[0044] Construction of recombinant Escherichia coli: First, the E. coli BL21(DE3) (Invitrogen) competent cells stored at -80 °C were ice-bathed at 0 °C for 10 min, then 5 μL of the recombinant product was added in a laminar flow hood, ice-bathed at 0 °C for 30 min, heat-shocked in a 42 °C water bath for 90 s, ice-bathed at 0 °C for 2 min, 600 μL of LB liquid medium was added, and cultured in a shaker at 37 °C and 200 rpm for 1 h; spread on an LB plate containing 50 μg / mL kanamycin resistance and cultured at 37 °C for 8 - 12 h, randomly pick clones to extract plasmids for sequencing identification, and screen to obtain recombinant Escherichia coli containing the expression recombinant plasmid E.coli BL21(DE3) / pET28a - BpSPase.

[0045] Example 2

[0046] Induced expression of sucrose phosphorylase:

[0047] Wet bacterial cells containing the sucrose phosphorylase gene: The recombinant Escherichia coli E.coli BL21(DE3) / pET28a - EfSPase obtained in Example 1 was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance, cultured at 37 °C and 200 rpm for 12 h, and then inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin resistance at an inoculation amount of 1% (v / v), and cultured at 37 °C and 200 rpm until the bacterial cells reached OD 600Reach 0.6 - 0.8. Add isopropyl - β - D - thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, induce and culture at 25 °C for 16 h, then centrifuge at 4 °C and 8000 rpm for 20 min, discard the supernatant, collect the precipitate, and obtain the wet cells of the recombinant strain pET28a - BpSPase containing sucrose phosphorylase.

[0048] Example 3

[0049] Establishment of the sucrose phosphorylase gene mutation library:

[0050] Using the E.coli BL21(DE3) / pET28a - BpSPase constructed in Example 2 as the starting strain.

[0051] Modify it through the method of directed evolution theory. According to the calculation of the protein language model, select the mutation sites W91A, R94P, V154T, H185G, G197A, T275L, Q301P, R304P, A323H, S352D, N424L for site - directed mutagenesis. The primers for each site - directed mutagenesis are shown in Table 1.

[0052] The mutation PCR system (100 μL) is: 25 μL of 2×Phanta Max buffer, 1 μL of dNTPs, 1 μL of each of the upstream and downstream mutation primers, 1 μL of the template (starting strain), 0.5 μL of Pfu DNA polymerase, and make up to 50 μL with ddH2O. The PCR conditions are: pre - denaturation at 95 °C for 3 min, followed by 30 cycles: 95 °C for 15 s, 60 °C for 15 s, 72 °C for 7 min 20 s, and finally a final extension at 72 °C for 10 min. The PCR products are respectively verified by DNA agarose gel electrophoresis for positivity. Digest the PCR products with DpnI enzyme for the template at 37 °C for 1 h, 200 rpm, inactivate at 65 °C for 1 minute, heat - shock transform the PCR products, and activate Escherichia coli E. coli BL21(DE3), place it at 37 °C, 200 rpm, culture for 1 h, spread it on an LB plate containing 50 μg / mL kanamycin resistance, and incubate it upside - down at 37 °C overnight.

[0053] Table 1 Design of primers for site - directed mutagenesis of sucrose phosphorylase

[0054]

[0055] Example 4

[0056] Screening of the sucrose phosphorylase gene mutation library:

[0057] Pick monoclonal colonies from the flat plate obtained in Example 3 and inoculate them into LB liquid medium containing 50 μg / mL kanamycin resistance. Culture at 37 °C and 200 rpm for 12 h. Preserve the bacterial strain and send it to a sequencing company for sequencing verification. After correct sequencing verification, inoculate the preserved bacterial strain into LB liquid medium containing 50 μg / mL kanamycin resistance at an inoculation amount of 0.2%. Culture at 37 °C and 200 rpm for 12 h, and then inoculate it into fresh LB liquid medium containing 50 μg / mL kanamycin resistance at an inoculation amount of 1% (v / v). Culture at 37 °C and 200 rpm until the OD 600 reaches 0.6 - 0.8. Add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, and induce culture at 25 °C for 16 h. Then centrifuge at 4 °C and 8000 rpm for 20 min, discard the supernatant, and collect the precipitate to obtain the wet bacterial cells containing the sucrose phosphorylase gene mutation library.

[0058] 1. Primary screening:

[0059] Prepare the reaction solution (200 μL): substrate sucrose with a final concentration of 200 mM, vitamin C with a final concentration of 180 mM, catalyst dosage 5 g / L based on the total weight of the wet bacterial cells, use pure water as the reaction medium, and adjust the pH of the reaction system to 5.0 with 1 M NaOH. Reaction conditions: React in the dark on a reactor at 40 °C and 500 rpm for 2 h. After the reaction ends, take 20 μL of the sample after the reaction, dilute it 200 times, filter it through a 0.22 μm filter membrane, and perform HPLC detection. The detection results are shown in Table 2.

[0060] Table 2 Primary screening reaction results

[0061]

[0062] 2. Secondary screening:

[0063] Perform secondary screening on the strains obtained from the primary screening. Combine and mutate the strains and send them to a sequencing company for sequencing verification. After correct sequencing verification, perform activity verification. Prepare the reaction solution (10 mL) for secondary screening: substrate sucrose with a final concentration of 500 mM, vitamin C with a final concentration of 450 mM, catalyst dosage 5 g / L based on the total weight of the wet bacterial cells, use pure water as the reaction medium, and adjust the pH of the reaction system to 5.0 with 1 M NaOH. Reaction conditions: React in the dark on a reactor at 40 °C and 500 rpm for 2 h. After the reaction ends, take 20 μL of the sample after the reaction, dilute it 200 times, filter it through a 0.22 μm filter membrane, and perform HPLC detection. The detection results are shown in Table 3.

[0064] Table 3 Secondary screening reaction results

[0065]

[0066] 3. Stability test:

[0067] The obtained combined mutant BpSPase-W91A-V154T-H185G-A323H-N424L was compared with the original BpSPase in terms of stability. The combined mutant and the crude enzyme solution of the original strain (with the same wet cell concentration of 50 g / L before disruption) were placed in a 40 °C water bath, and samples were taken at different times for enzyme activity detection. Detection system (10 mL): The final concentration of the substrate sucrose was 500 mM, the final concentration of vitamin C was 450 mM, the amount of crude enzyme solution used was 1 mL, pure water was used as the reaction medium, and the pH of the reaction system was adjusted to 5.0 with 1 M NaOH. Reaction conditions: After reacting for 2 h in the dark on a reactor at 40 °C and 500 rpm, 20 μL of the sample at the end of the reaction was taken, diluted 200 times, filtered through a 0.22 μm filter membrane, and subjected to HPLC detection. The detection results are shown in Table 4. From the results, the stability of the mutant BpSPase-W91A-V154T-H185G-A323H-N424L was significantly better than that of the original strain.

[0068] Table 4 Stability reaction results

[0069]

[0070] Example 5

[0071] Application of sucrose phosphorylase in the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid:

[0072] The recombinant sucrose phosphorylase mutant with the highest activity obtained in Example 4 E.coli BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L was inoculated into an LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and cultured at 37 °C for 9 h as the seed solution. It was then inoculated into a 50 L fermenter containing 30 L of fermentation medium at an inoculation volume concentration of 3.5%. It was cultured at 37 °C and 500 rpm for about 3 - 4 h until the cell density OD reached 6 - 8. After the temperature of the fermenter was lowered to 25 °C, lactose at a final concentration of 10 g / L was added as an inducer, and then it was cultured at 25 °C and 500 rpm for 12 h. The cultured fermentation broth was centrifuged at 8000 rpm for 10 min to obtain the sucrose phosphorylase mutant-containing E.coliWet cells of BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L.

[0073] The composition of the fermentation tank medium: 450 g of tryptone, 360 g of yeast extract, 300 g of sodium chloride, 40.8 g of potassium dihydrogen phosphate, 450 g of glycerol, 68.4 g of dipotassium hydrogen phosphate trihydrate, 150 g of ammonium sulfate, 11.25 g of magnesium sulfate, 30 g of antifoaming agent. Add distilled water to make up the volume to 30 L and dissolve.

[0074] The dosage of the catalyst is 20 g / L based on the total weight of the wet cells, and the final concentration is 1.3 M (sucrose is added in two portions, 0.65 M of sucrose is added at the beginning of the reaction, and the remaining 0.65 M of sucrose is added after the reaction proceeds for 3 h). The substrate sucrose has a final concentration of 1.2 M, the substrate vitamin C has a final concentration of 1.2 M, pure water is used as the reaction medium, and the pH of the reaction system is adjusted to 5.0 with 1 M NaOH. The total volume of the reaction solution is 10 L. Reaction conditions: 40 °C, 300 rpm, reaction in the dark for 15 h. After the reaction is completed, 20 μL of the reaction sample is taken, diluted 500 times, filtered through a 0.22 μm filter membrane, and detected by HPLC. The reactions occurring during the catalysis are as Figure 1 shown, and the reaction progress curve is as Figure 2 shown. After the reaction is completed, the concentration of 2-O-α-D-glucopyranosyl-L-ascorbic acid is 358 g / L (1.05 M).

[0075] In another embodiment, the dosage of the catalyst is 5 g / L based on the total weight of the wet cells, the final concentration of the substrate sucrose is 1.2 M, the final concentration of the substrate vitamin C is 1.1 M, pure water is used as the reaction medium, the pH of the reaction system is 5.0, and the total volume of the reaction solution is 10 L.

[0076] In another embodiment, the dosage of the catalyst is 25 g / L based on the total weight of the wet cells, the final concentration of the substrate sucrose is 1.4 M, the final concentration of the substrate vitamin C is 1.3 M, pure water is used as the reaction medium, the pH of the reaction system is 5.0, and the total volume of the reaction solution is 10 L.

[0077] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A sucrose phosphorylase mutant, characterized in that, The wild-type sucrose phosphorylase with the amino acid sequence shown in SEQ ID NO.2 has the 91st tryptophan mutated to alanine, the 154th valine mutated to threonine, the 185th histidine mutated to glycine, the 323rd alanine mutated to histidine, and the 424th asparagine mutated to leucine.

2. A gene encoding the sucrose phosphorylase mutant according to claim 1.

3. An expression cassette, characterized in that Comprising the gene according to claim 2.

4. A recombinant expression vector, characterized in that, Comprising the gene according to claim 2.

5. A transgenic cell line, characterized in that, Comprising the gene according to claim 2.

6. A recombinant bacterium, characterized in that, Comprising the gene according to claim 2.

7. Use of the recombinant bacterium according to claim 6 in the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

8. A method for synthesizing 2-O-α-D-glucopyranosyl-L-ascorbic acid, characterized in that, Including: Using the wet cells obtained by induced culture of the genetically engineered bacterium of the sucrose phosphorylase mutant, or the crude enzyme solution extracted by ultrasonic disruption of the wet cells, or the immobilized enzyme as a catalyst, using sucrose and vitamin C as substrates, and pure water as a reaction medium to form a reaction system, and reacting to obtain 2-O-α-D-glucopyranosyl-L-ascorbic acid; Wherein, the genetically engineered bacterium is constructed by introducing the sucrose phosphorylase mutant according to claim 1 into a host bacterium.

9. The method according to claim 8, characterized in that, The dosage of the catalyst is 5 g / L to 25 g / L based on the total weight of the wet cells.

10. The method according to claim 8, wherein The final concentration of the substrate sucrose is 1.2 M to 1.4 M, and the final concentration of the substrate vitamin C is 1.1 M to 1.3 M.

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