Sucrose phosphorylase mutant and application thereof in synthesis of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a sucrose phosphorylase mutant and application thereof in synthesis of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid. The sucrose phosphorylase mutant is obtained by simultaneously mutating multiple sites of amino acids at the 91st site, the 154th site, the 185th site, the 323rd site and the 424th site of wild sucrose phosphorylase derived from Bifidobacterium pseudolongum, so that the efficiency of producing 2-O-alpha-D-glucopyranosyl-L-ascorbic acid by taking sucrose and vitamin C as raw materials is remarkably improved; only 15 hours are needed for completely catalyzing 1.2 M of vitamin C to produce 2-O-alpha-D-glucopyranosyl-L-ascorbic acid; and the yield of the 2-O-alpha-D-glucopyranosyl-L-ascorbic acid reaches 358 g / L. Therefore, the 2-O-alpha-D-glucopyranosyl-L-ascorbic acid has an important application value in the field of production of the 2-O-alpha-D-

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 overall understanding of the present invention, and it is not necessarily regarded as an admission or an implication in any form 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 easy oxidation of the hydroxyl group at the C2 position of L-ascorbic acid, 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 than L-ascorbic acid in an environment where oxygen, heat, and metal ions are present. 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 skin or in vivo enzymes (such as α-glucosidase) to slowly release vitamin C and continuously exert 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 enzymatic methods, using enzymes to catalyze the reaction of vitamin C with glycosyl donors, with mild conditions and environmental friendliness; it can also be synthesized chemically, but chemical synthesis has problems such as more impurities, complex purification processes, many impurities, and serious pollution. Therefore, at present, mainly highly specific enzymatic methods are used 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 reported sucrose phosphorylase and its mutants currently have problems of 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, a sucrose phosphorylase mutant is provided, which is obtained by simultaneous multi-site mutations 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 a mutant in which 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, a gene encoding the sucrose phosphorylase mutant described in the first aspect is provided.

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

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

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

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

[0013] In the seventh aspect of the present invention, the application of the sucrose phosphorylase mutant described in the first aspect, 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 is provided.

[0014] In the eighth aspect of the present invention, a method for synthesizing 2-O-α-D-glucopyranosyl-L-ascorbic acid is provided, 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 described in the first aspect into a host bacterium.

[0015] 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 bacterial 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.

[0016] The beneficial effects of the present invention are as follows: (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.

[0017] (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

[0018] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0019] Figure 1 is E.coli The reaction formula for the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid by BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L; Figure 2 is E.coli The reaction process diagram for the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid by BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L. Detailed Embodiments

[0020] It should be noted that the following detailed description is exemplary and is intended to provide further explanation 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 belongs.

[0021] 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 form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] 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.

[0023] The medium formulations used in the following examples are as follows: LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is water, and the pH is 7.4.

[0024] LB plate: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, 18 g / L agar, the solvent is water, and the pH is 7.4.

[0025] 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: Column model: QS-C18, 5 μm, 4.6×250 mm; the mobile phase is 20 mM potassium dihydrogen phosphate solution, adjusted to pH = 2 with phosphoric acid, the injection volume is 10 μL; ultraviolet detector; detection wavelength: 242 nm; detection time: 10 min; flow rate: 0.8 mL / min; column temperature: 30 °C.

[0026] 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.

[0027] Example 1 Construction of expression vector and engineering bacteria: 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 synthesized by GenScript Biotech Corporation in Nanjing, and its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO:2.

[0028] 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.

[0029] F1: 5’-ctttaagaaggagatataccATGAAAAATAAGGTACAACTAATAACATATGC-3’; R1: 5’-tggtggtggtggtgctcgagTTAATCCATGTGTGCTACCGGC-3’; F2: 5’-CTCGAGCACCACCACCACC-3’; R2: 5’-GGTATATCTCCTTCTTAAAGTTAAACAAAAT-3’; Using the pET-28a plasmid as the expression vector, Escherichia coli E.coli BL21(DE3) / pET-28a-BpSPase was constructed.

[0030] Construction of the expression plasmid: Under the initiation of primers F1 / R1 and F2 / R2, using the target gene as the template, the sucrose phosphorylase gene sequence with homologous arms was amplified using high-fidelity Pfu DNA polymerase. Using the pET-28a plasmid as the 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 homologous recombinase to construct the plasmid pET28a-BpSPase.

[0031] Preparation of competent cells: Obtain the glycerol stock of E. coli BL21(DE3) strain from the -80 °C refrigerator, streak it on an antibiotic-free LB plate, and culture it at 37 °C for 10 h to obtain single colonies; pick a single colony from the LB plate and inoculate it into a test tube containing 5 mL of LB liquid medium, and culture it at 37 °C and 180 rpm for 9 h; take 200 μL of the bacterial solution from the test tube and inoculate it into 50 mL of LB liquid medium, and culture it at 37 °C and 180 rpm until the OD 600To 0.4 - 0.6; Pre - cool the bacterial solution on ice. Take the bacterial solution into a sterilized centrifuge tube, place it on ice for 10 min, centrifuge at 4 °C and 5000 rpm for 10 min; Pour out the supernatant, being careful to prevent contamination. Resuspend the precipitated cells with pre - cooled 0.1 mol / L CaCl₂ aqueous solution and place it on ice for 30 min; Centrifuge at 4 °C and 5000 rpm for 10 min, discard the supernatant, resuspend the precipitated cells with pre - cooled 0.1 mol / L CaCl₂ aqueous solution containing 15% glycerol. Take 100 μL of the resuspended cells and aliquot them into sterilized 1.5 mL centrifuge tubes, store them in a - 80 °C refrigerator, and take them out when needed.

[0032] Construction of recombinant Escherichia coli: First, thaw the E. coli BL21(DE3) (Invitrogen) competent cells stored at - 80 °C in an ice bath at 0 °C for 10 min, then add 5 μL of the recombinant product in a laminar flow hood, ice - bath at 0 °C for 30 min, heat - shock in a 42 °C water bath for 90 s, ice - bath at 0 °C for 2 min, add 600 μL of LB liquid medium, culture in a shaker at 37 °C and 200 rpm for 1 h; Spread on an LB plate containing 50 μg / mL kanamycin resistance and culture at 37 °C for 8 - 12 h. Randomly pick colonies, extract plasmids for sequencing and identification, and screen to obtain recombinant Escherichia coli containing the expression recombinant plasmid E.coli BL21(DE3) / pET28a - BpSPase.

[0033] Example 2 Induced expression of sucrose phosphorylase: Wet bacterial cells containing the sucrose phosphorylase gene: Resuspend the recombinant Escherichia coli E.coli BL21(DE3) / pET28a - EfSPase obtained in Example 1 into an LB liquid medium containing 50 μg / mL kanamycin resistance, culture at 37 °C and 200 rpm for 12 h, then inoculate 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 cell OD 600 reaches 0.6 - 0.8, add isopropyl - β - D - thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, 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 of the recombinant strain pET28a - BpSPase containing sucrose phosphorylase.

[0034] Example 3 Establishment of a sucrose phosphorylase gene mutation library: Using the one constructed in Example 2E.coli BL21(DE3) / pET28a-BpSPase was used as the starting strain.

[0035] It was modified by the method of directed evolution theory. According to the calculation of the protein language model, the mutation sites W91A, R94P, V154T, H185G, G197A, T275L, Q301P, R304P, A323H, S352D, and N424L were selected for site-directed mutagenesis. The site-directed mutagenesis primers are shown in Table 1.

[0036] The mutation PCR system (100 μL) was as follows: 25 μL of 2×Phanta Max buffer, 1 μL of dNTPs, 1 μL each of the forward and reverse mutation primers, 1 μL of the template (starting strain), 0.5 μL of Pfu DNA polymerase, and ddH2O was added to make up to 50 μL. The PCR conditions were: 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 were verified by DNA agarose gel electrophoresis. The PCR products were digested with DpnI enzyme for the template at 37 °C for 1 h, 200 rpm, inactivated at 65 °C for 1 minute, and the PCR products were heat-shock transformed. Then Escherichia coli E. coli BL21(DE3) was activated, incubated at 37 °C, 200 rpm for 1 h, spread on an LB plate containing 50 μg / mL kanamycin resistance, and incubated upside down at 37 °C overnight.

[0037] Table 1 Design of site-directed mutagenesis primers for sucrose phosphorylase

[0038] Example 4 Screening of the sucrose phosphorylase gene mutation library: Single colonies were picked from the plate obtained in Example 3 and inoculated into an LB liquid medium containing 50 μg / mL kanamycin resistance, incubated at 37 °C, 200 rpm for 12 h. The preserved strains were sent to a sequencing company for sequencing verification. After correct sequencing verification, the preserved strains were inoculated into an LB liquid medium containing 50 μg / mL kanamycin resistance at an inoculation amount of 0.2%, incubated at 37 °C, 200 rpm for 12 h, and then inoculated into a fresh LB liquid medium containing 50 μg / mL kanamycin resistance at an inoculation amount of 1% (v / v), and incubated at 37 °C, 200 rpm until the cell OD 600Reach 0.6 - 0.8. Add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mM. After inducing culture at 25 °C for 16 h, 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.

[0039] 1. Primary screening: Prepare the reaction solution (200 μL): Substrate sucrose with a final concentration of 200 mM, vitamin C with a final concentration of 180 mM, the catalyst dosage is 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 at 40 °C and 500 rpm in a reactor for 2 h. After the reaction ends, take 20 μL of the sample at the end of 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.

[0040] Table 2 Primary screening reaction results

[0041] 2. Secondary screening: Perform secondary screening on the strains obtained from the primary screening. Combine and mutate the mutants 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, the catalyst dosage is 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 at 40 °C and 500 rpm in a reactor for 2 h. After the reaction ends, take 20 μL of the sample at the end of 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.

[0042] Table 3 Secondary screening reaction results

[0043] 3. Stability test: 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 reaction 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.

[0044] Table 4 Stability reaction results

[0045] Example 5 Application of sucrose phosphorylase in the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid: The recombinant sucrose phosphorylase mutant with the highest activity obtained in Example 4 E.coli BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L. It was inoculated into an LB liquid medium containing kanamycin with a final concentration of 50 μg / mL and cultured at 37 °C for 9 h as the seed solution. Then, it was 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 with 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 wet cells containing the sucrose phosphorylase mutant E.coli BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L.

[0046] The composition of the fermentation broth 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 (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 a constant volume of 30 L for dissolution.

[0047] 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: React in the dark at 40 °C and 300 rpm for 15 h. After the reaction is completed, take 20 μL of the sample at the end of the reaction, dilute it 500 times, filter it through a 0.22 μm filter membrane, and perform HPLC detection. 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).

[0048] 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.

[0049] 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.

[0050] 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 modifications, equivalent replacements, improvements, 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 91st tryptophan of wild-type sucrose phosphorylase with the amino acid sequence shown in SEQ ID NO.2 is mutated to alanine, the 154th valine is mutated to threonine, the 185th histidine is mutated to glycine, the 323rd alanine is mutated to histidine, and the 424th asparagine is 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 the 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, wherein 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, characterized in that, 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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