An immobilized cell, a preparation method thereof, and a preparation method of 2-O-α-D-glucopyranosyl-L-ascorbic acid

Abstract

The present invention belongs to the field of bioengineering technology, and particularly relates to an immobilized cell, a preparation method thereof, and a preparation method of 2-O-α-D-glucopyranosyl-L-ascorbic acid. The present invention immobilizes a sucrose phosphorylase mutant cell derived from Bifidobacterium pseudolongum ( Bifidobacterium pseudolongum ), thereby significantly improving its stability, acid resistance, and reusability. Using sucrose and vitamin C as raw materials, it produces 2-O-α-D-glucopyranosyl-L-ascorbic acid, and its activity remains above 90% after continuous use for 10 batches. It has great application value in the production field of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to an immobilized cell, a preparation method thereof, and a preparation method 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 an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a glycoside derivative of L-ascorbic acid (L-AA, also known as vitamin C) with significantly improved stability. This compound and its preparation technology were first jointly developed by the Chemical Research Institute of Hayashibara Co., Ltd. in Japan and the Department of Pharmacy of Okayama University in Japan in 1990. It is obtained by substituting the C2 hydroxyl group of L-ascorbic acid with a glucosyl group. Since C2 and C3 in the L-ascorbic acid structure are enediol structures, it has extremely strong reducibility and is extremely unstable under conditions such as oxygen, metal ions, light, and alkalinity, which limits its industrial application. However, 2-O-α-D-glucopyranosyl-L-ascorbic acid itself does not have reducibility and is not prone to oxidation reactions. It has better stability compared with L-ascorbic acid. Moreover, through the hydrolysis of α-glucosidase, it generates L-ascorbic acid and glucose, exerting the same reducibility and antioxidant properties as L-ascorbic acid. At the same time, it also extends the action duration of L-ascorbic acid. Therefore, it is a good substitute for L-ascorbic acid.

[0004] The prior art provides the application of sucrose phosphorylase or its mutants in the production process of 2-O-α-D-glucopyranosyl-L-ascorbic acid. The production technology using sucrose phosphorylase will produce by-products with structures similar to 2-O-α-D-glucopyranosyl-L-ascorbic acid and with stability inferior to 2-O-α-D-glucopyranosyl-L-ascorbic acid, which are difficult to remove in the downstream separation and purification process; the existing technical solutions based on mutants have higher conversion rates of L-ascorbic acid and higher product purity of 2-O-α-D-glucopyranosyl-L-ascorbic acid. In order to improve economic efficiency, the whole cells after the reaction are separated and sucrose and vitamin C are used again as substrates to produce 2-O-α-D-glucopyranosyl-L-ascorbic acid, and it is found that these cells have almost no activity. Summary of the Invention

[0005] To overcome the above-mentioned problems and improve economic benefits, the present invention provides immobilized cells, a method for preparing the same, and a method for preparing 2-O-α-D-glucopyranosyl-L-ascorbic acid. Through cell immobilization, the cells can be reused as a catalyst in multiple batches, which has extremely high application value for the industrial production of 2-O-α-D-glucopyranosyl-L-ascorbic acid. To achieve the above-mentioned goals, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a method for preparing immobilized cells, wherein the cells are immobilized using an embedding-crosslinking method, specifically using sodium alginate for embedding and polyethyleneimine-glutaraldehyde for composite crosslinking; the method comprises the following steps:

[0007] S1, preparing microspheres: suspending wet cells of the sucrose phosphorylase mutant in water, mixing with a sodium alginate solution, then adding carboxymethyl chitosan and cellulose nanocrystals, and continuing to stir thoroughly to obtain a mixed solution, then dropping the mixed solution into a CaCl2 solution and solidifying it by magnetic stirring; collecting the microspheres;

[0008] In one or more embodiments of the present invention, the nucleotide sequence of the sucrose phosphorylase mutant is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2;

[0009] S2, polyethyleneimine-genipin composite cross-linking: adding the microspheres obtained in S1 to a polyethyleneimine aqueous solution, magnetically stirring, and then collecting the microspheres; then adding the microspheres to the genipin solution, magnetically stirring, and then collecting the microspheres to obtain S2.

[0010] In step S1, the mass ratio of wet bacteria to sodium alginate (m / m) is 1:0.5-1:2, the optimal ratio is 1:1, the mass ratio of wet bacteria to carboxymethyl chitosan (m / m) is 1:0.5-1:2, the optimal ratio is 1:1, and the mass ratio of wet bacteria to cellulose nanocrystals (m / m) is 15:3-15:15, and the optimal ratio is 15:8.

[0011] In step S2, the mass ratio (m / m) of microspheres to polyethyleneimine is 15:1-5:1, and the optimal ratio is 10:1. The mass ratio (m / m) of microspheres to genipin is 15:1-5:1, and the optimal ratio is 10:1.

[0012] In the preparation method provided by the present invention, the addition of carboxymethyl chitosan can improve the cell embedding effect, and the addition of cellulose nanocrystals can make the immobilized microspheres stronger and less prone to breakage.

[0013] The present invention improves the cross-linking effect through composite cross-linking; through comparative experiments, it is found that the stability of immobilized cells obtained by polyethyleneimine-genipin composite cross-linking is better than that of other composite cross-linking schemes.

[0014] In the second aspect of the present invention, there is provided an immobilized cell of a genetically engineered bacterium of a sucrose phosphorylase mutant prepared by the method for preparing the immobilized cell.

[0015] In the third aspect of the present invention, there is provided a recombinant sucrose phosphorylase bacterium comprising the immobilized cell of the genetically engineered bacterium of the sucrose phosphorylase mutant described in the second aspect.

[0016] In the fourth aspect of the present invention, there is provided the use of the immobilized cell of the genetically engineered bacterium of the sucrose phosphorylase mutant described in the second aspect or the recombinant sucrose phosphorylase bacterium described in the third aspect in the preparation of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

[0017] In the fifth aspect of the present invention, there is provided a method for preparing 2-O-α-D-glucopyranosyl-L-ascorbic acid, comprising:

[0018] Using the immobilized genetically engineered bacterium cells of the sucrose phosphorylase mutant 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;

[0019] wherein, the immobilized genetically engineered bacterium cells of the sucrose phosphorylase mutant are the recombinant sucrose phosphorylase bacterium described in the third aspect.

[0020] In one or more embodiments, the dosage of the catalyst is 5 g / L to 45 g / L based on the total weight of the immobilized 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.

[0021] The beneficial effects of the present invention are as follows:

[0022] In the present invention, the immobilized cells of the sucrose phosphorylase mutant derived from Bifidobacterium pseudolongum ( Bifidobacterium pseudolongum ) are immobilized, thereby significantly improving its stability, acid resistance, and reusability. As a catalyst, using sucrose and vitamin C as raw materials to produce 2-O-α-D-glucopyranosyl-L-ascorbic acid, the activity remains above 90% after continuous use for 10 batches. It has great economic benefits and application value in the production field of 2-O-α-D-glucopyranosyl-L-ascorbic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0024] 图1 For immobilization E.coli 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. Specific implementation manners

[0025] 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 belongs.

[0026] It should be noted that the terms used herein are only for describing specific implementation manners 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 indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] 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.

[0028] The medium formulations used in the following examples are as follows:

[0029] LB liquid medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, the solvent is water, and the pH is 7.4.

[0030] Fermenter medium: 15 g / L of tryptone, 12 g / L of yeast extract, 10 g / L of sodium chloride, 1.36 g / L of potassium dihydrogen phosphate, 15 g / L of glycerol, 2.28 g / L of dipotassium hydrogen phosphate trihydrate, 5 g / L of ammonium sulfate, 0.375 g / L of magnesium sulfate, 1 g / L of antifoaming agent, and distilled water is added for dissolution.

[0031] 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:

[0032] Chromatographic column model: QS-C18, 5 μm, 4.6×250 mm; mobile phase: 20 mM potassium dihydrogen phosphate solution, adjusted to pH = 2 with phosphoric acid, injection volume: 10 μL; UV detector; detection wavelength: 242 nm; detection time: 10 min; flow rate: 0.8 mL / min; column temperature: 30 °C.

[0033] Sample treatment: Take 5 μL of the sample after the reaction is completed, dilute it 600 times with aqueous solution, filter it through a 0.22 μm filter membrane, and perform HPLC detection.

[0034] Example 1, culture and fermentation of strains

[0035] Early mining originated from Bifidobacterium pseudolongum ( Bifidobacterium pseudolongum ) sucrose phosphorylase (BpSPase, NCBI accession number WP_129853343.1), a mutant was obtained by transformation E.coli BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L (nucleotide sequence is shown in SEQ ID NO. 1, amino acid sequence is shown in SEQ ID NO. 2).

[0036] Recombinant sucrose phosphorylase mutant E.coli BL21 (DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37 ° C for 9 hours. As a seed solution, it was inoculated into a 50 L fermenter containing 30 L fermenter medium at a volume concentration of 3.5%. Cultivate at 37 ° C, 500 rpm for about 3-4 hours. After the bacterial density reached OD 6-8, the fermenter temperature was lowered to 25 ° C, and lactose was added as an inducer at a final concentration of 10 g / L. Then, cultured at 25 ° C, 500 rpm for 12 hours. The fermented broth was centrifuged at 8000 rpm for 10 minutes to obtain the sucrose phosphorylase mutant. E.coli Wet cells of BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L.

[0037] Example 2, Immobilization of Cells

[0038] Experimental group:

[0039] Cell immobilization was carried out by the embedding-crosslinking method. Sodium alginate embedding: The wet bacterial cells harvested by centrifugation in Example 1 were washed twice with pure water, suspended in pure water (30 g / L), mixed with a 3% (m / V, 30 g of sodium alginate added to every 1000 mL of water) sodium alginate solution of the same volume as the bacterial suspension, stirred thoroughly for 20 min, then 1.5% (m / V, V is the volume of the mixed solution of the bacterial suspension and the sodium alginate solution) carboxymethyl chitosan and 0.8% (m / V, V is the volume of the mixed solution of the bacterial suspension and the sodium alginate solution) cellulose nanocrystals were added, and stirring was continued thoroughly for 20 min. Then, the mixed solution was slowly dropped into 0.1 M CaCl2 solution at a rate of 100 drops / min through a dropping device, and magnetically stirred (100 rpm) for 1 h. Then, all the microspheres were collected with a sieve (pore size <1 mm) to obtain microspheres containing the sucrose phosphorylase mutant E. coli BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L, and washed 3 times with pure water.

[0040] Polyethyleneimine-genipin composite crosslinking (the composite crosslinking agent can make the crosslinking effect between the microspheres and the crosslinking agent better): The embedded microspheres were added to a 0.3% (m / V) polyethyleneimine aqueous solution at 30 g / L, magnetically stirred (100 rpm) for 1 h, then all the microspheres were collected with a sieve (pore size <1 mm) and washed 3 times with pure water. Then, the microspheres were added to a 0.3% (m / V) genipin solution at 30 g / L, magnetically stirred (100 rpm) for 4 h, then all the microspheres were collected with a sieve (pore size <1 mm) and washed 3 times with pure water. They were stored in sterile water at 4°C for later use.

[0041] Control group 1:

[0042] Cell immobilization was carried out by the embedding-crosslinking method. Sodium alginate embedding: The wet bacterial cells harvested by centrifugation in Example 1 were washed twice with pure water, suspended in pure water (30 g / L), mixed with a 3% (m / V) sodium alginate solution of the same volume as the bacterial suspension, stirred thoroughly for 20 min, then the mixed solution was slowly dropped into 0.1 M CaCl2 solution at a rate of 100 drops / min through a dropping device, and magnetically stirred (100 rpm) for 1 h. Then, all the microspheres were collected with a sieve (pore size <1 mm) to obtain microspheres containing the sucrose phosphorylase mutant E. coli BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L, and washed 3 times with pure water.

[0043] Polyethyleneimine-glutaraldehyde composite crosslinking: Polyethyleneimine-glutaraldehyde composite crosslinking: The embedded alginate microspheres were added to a 0.3% (m / V) polyethyleneimine solution, and magnetically stirred (100 rpm) for 1 h. Then all the microspheres were collected with a sieve (pore size <1 mm) and washed 3 times with pure water. Subsequently, the microspheres were added to a 1% (V / V) glutaraldehyde solution, magnetically stirred (100 rpm) for 5 min, then all the microspheres were collected with a sieve (pore size <1 mm) and washed 3 times with pure water. They were stored in sterile water at 4 °C for later use.

[0044] Control group 2:

[0045] Cell immobilization was carried out by the embedding-crosslinking method. Chitosan embedding: The wet bacterial cells harvested by centrifugation in Example 1 were washed 2 times with pure water. The wet bacterial cells were suspended in pure water (30 g / L). 5% (m / V) chitosan equal in volume to the bacterial suspension was dissolved in 1% (V / V) acetic acid solution. The solution was mixed well and stirred vigorously for 20 min. Then the mixture was slowly dropped into 0.2 M NaOH solution at a rate of 100 drops / min through a dropping device and magnetically stirred (100 rpm) for 5 min. Then all the microspheres containing the sucrose phosphorylase mutant E. coli BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L were collected with a sieve (pore size <1 mm) and washed 3 times with pure water.

[0046] Polyethyleneimine-genipin composite crosslinking: The embedded microspheres were added to a 0.3% (m / V) aqueous polyethyleneimine solution, and magnetically stirred (100 rpm) for 1 h. Then all the microspheres were collected with a sieve (pore size <1 mm) and washed 3 times with pure water. Subsequently, the microspheres were added to a 0.3% (m / V) genipin solution, magnetically stirred (100 rpm) for 4 h, then all the microspheres were collected with a sieve (pore size <1 mm) and washed 3 times with pure water. They were stored in sterile water at 4 °C for later use.

[0047] Control group 3:

[0048] Cell immobilization was carried out by the embedding-crosslinking method. Sodium alginate embedding: The wet bacterial cells harvested by centrifugation in Example 1 were washed twice with pure water, suspended in pure water (30 g / L), mixed with an equal volume of 5% (m / V) sodium alginate solution, stirred thoroughly for 20 min, then 1.5% (m / V, V is the volume of the mixture of the bacterial suspension and the sodium alginate solution) carboxymethyl chitosan and 1% (m / V, V is the volume of the mixture of the bacterial suspension and the sodium alginate solution) cellulose nanocrystals were added, and stirring was continued thoroughly for 20 min. Then, the mixture was slowly dropped into 0.1 M CaCl2 solution at a rate of 100 drops / min through a dropping device and magnetically stirred (100 rpm) for 1 h. Then all the microspheres were collected with a sieve to obtain microspheres containing the sucrose phosphorylase mutant E. coli BL21(DE3) / pET28a-BpSPase-W91A-V154T-H185G-A323H-N424L, and washed 3 times with pure water.

[0049] Polyethyleneimine-genipin composite crosslinking (the composite crosslinking agent can make the crosslinking effect between the microspheres and the crosslinking agent better): The embedded microspheres were added to 0.5% (m / V) polyethyleneimine aqueous solution at 30 g / L, magnetically stirred (100 rpm) for 1 h, then all the microspheres were collected with a sieve and washed 3 times with pure water. Then the microspheres were added to 0.5% (m / V) genipin solution at 30 g / L, magnetically stirred (100 rpm) for 4 h, then all the microspheres were collected with a sieve and washed 3 times with pure water. They were stored at 4°C in sterile water for later use.

[0050] Example 3, Application of immobilized cells in the catalytic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid

[0051] Immobilized 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 is as 图1 shown.

[0052] The amount of catalyst is 40 g / L based on the total weight of immobilized cells, with a final concentration of 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 0.6 M of sucrose is added after the reaction proceeds for 3 h). The substrate is sucrose with a final concentration of 1.2 M, and the substrate is vitamin C with a final concentration of 1.2 M. Pure water is used as the reaction medium. The pH of the reaction system is adjusted to 5.0 with 1 M NaOH, and the total volume of the reaction solution is 1 L. Reaction conditions: React at 40 °C and 300 rpm in the dark for 15 h. After the reaction ends, 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. Then collect the microspheres by sieving, and wash them 3 times with pure water. Then put them into the same reaction system and repeat the same steps. A total of 10 batches of reactions are carried out, and the detection results of each batch are shown in Table 1. From the results, it can be seen that the immobilized cells in the experimental group can be reused 10 batches, and the activity can still be maintained above 90%.

[0053] Table 1. Experimental results of the reuse of immobilized cells for 10 batches

[0054]

[0055] In another embodiment, the amount of catalyst is 5 g / L based on the total weight of immobilized cells, with a final concentration of 1.2 M of the substrate sucrose and a final concentration of 1.1 M of the substrate vitamin C. The pH of the reaction system is 5.0, and the total volume of the reaction solution is 1 L.

[0056] In another embodiment, the amount of catalyst is 45 g / L based on the total weight of immobilized cells, with a final concentration of 1.4 M of the substrate sucrose and a final concentration of 1.3 M of the substrate vitamin C. The pH of the reaction system is 5.0, and the total volume of the reaction solution is 1 L.

[0057] Example 4. Storage stability test of immobilized cells

[0058] To determine the stability of immobilized cells during storage, the activity of the immobilized cells stored at 4 °C is detected at regular intervals. The control group is free cells. The reaction solution (10 mL) of the activity detection system: the final concentration of the substrate sucrose is 500 mM, the final concentration of vitamin C is 450 mM, the amount of catalyst is 10 g / L based on the total weight of immobilized cells (the control group is the equivalent amount of free cells before immobilization), pure water is used as the reaction medium, and the pH of the reaction system is adjusted to 5.0 with 1 M NaOH. Reaction conditions: React at 40 °C and 500 rpm in the dark for 2 h on a reactor. 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. From the results, it can be seen that the storage stability of the immobilized sucrose phosphorylase mutant cells is significantly enhanced, and they can still maintain 73% of the activity after 25 days.

[0059] Table 2. Storage stability test results of immobilized cells

[0060]

[0061] Example 5, Temperature Stability Test of Immobilized Cells

[0062] To determine the temperature stability of immobilized cells, free and immobilized cells were placed in a 40°C or 50°C water bath for 12 hours, respectively. Samples were then taken for activity assays. The activity assay reaction solution (10 mL) consisted of a final concentration of 500 mM sucrose substrate, 450 mM vitamin C, and 10 g / L catalyst based on the total weight of immobilized cells (the control group consisted of an equivalent amount of free cells before immobilization). Purified water was used as the reaction medium, and the pH of the reaction system was adjusted to 5.0 with 1 M NaOH. The reaction conditions were: 40°C, 500 rpm, in a dark-protected reactor. After the reaction, 20 μL of the sample was diluted 200-fold, filtered through a 0.22 μm filter, and analyzed by HPLC. The results are shown in Table 3. The results indicate that the immobilized sucrose phosphorylase mutant cells exhibit significantly improved temperature stability compared to free cells.

[0063] Table 3. Experimental results of temperature stability test of immobilized cells

[0064]

[0065] Example 6, Acid resistance test of immobilized cells

[0066] In the reaction of producing 2-O-α-D-glucopyranosyl-L-ascorbic acid with sucrose and vitamin C catalyzed by sucrose phosphorylase, sucrose phosphorylase is quite sensitive to the reaction pH. In an overly acidic or alkaline environment, the activity of sucrose phosphorylase decreases significantly. Moreover, vitamin C is not suitable in an alkaline environment and is prone to oxidation. Therefore, the reaction pH should be slightly acidic. To determine the stability of immobilized cells in an acidic environment, free cells and immobilized cells were respectively placed in different pH systems for activity detection. The reaction solution (10 mL) of the activity detection system: the final concentration of the substrate sucrose was 500 mM, the final concentration of vitamin C was 450 mM, the dosage of the catalyst was 10 g / L based on the total weight of the immobilized cells (the control group was the equivalent amount of free cells before immobilization), pure water was used as the reaction medium, and the pH of the reaction system was adjusted to 3.0, 4.0, and 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 detected by HPLC. The detection results are shown in Table 4. From the results, the acid tolerance of the immobilized sucrose phosphorylase mutant cells was significantly improved compared with that of the free cells.

[0067] Table 4. Experimental results of the acid tolerance test of immobilized cells

[0068]

[0069] 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 changes and modifications. 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 method for preparing immobilized cells, characterized in that, It includes the following steps: S1. Preparation of microspheres: Suspend the wet cells of the sucrose phosphorylase mutant in water, mix it with the sodium alginate solution, then add carboxymethyl chitosan and cellulose nanocrystals, continue to stir well to obtain a mixed solution, and then drop the mixed solution into the CaCl2 solution and stir magnetically for solidification; collect the microspheres; in step S1, the nucleotide sequence of the sucrose phosphorylase mutant is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2; S2. Polyethyleneimine-genipin composite crosslinking: Add the microspheres obtained in S1 into the aqueous solution of polyethyleneimine, stir magnetically, and then collect the microspheres; then add the microspheres into the genipin solution, stir magnetically, and collect the microspheres again to obtain the product.

2. The immobilized cells of the genetically engineered bacterium of the sucrose phosphorylase mutant prepared by the method for preparing immobilized cells according to claim 1.

3. A recombinant bacterium of sucrose phosphorylase, characterized in that, It contains the immobilized cells of the genetically engineered bacterium of the sucrose phosphorylase mutant according to claim 2.

4. The application of the sucrose phosphorylase recombinant bacterium according to claim 3 in the preparation of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

5. A method for preparing 2-O-α-D-glucopyranosyl-L-ascorbic acid, characterized in that, It includes: Using the immobilized cells of the genetically engineered bacterium of the sucrose phosphorylase mutant 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; Among them, the immobilized cells of the genetically engineered bacterium of the sucrose phosphorylase mutant are the sucrose phosphorylase recombinant bacterium according to claim 3.

6. The preparation method of 2-O-α-D-glucopyranosyl-L-ascorbic acid according to claim 5, characterized in that, The dosage of the catalyst is 5 g / L to 45 g / L based on the total weight of the immobilized cells.

7. The preparation method of 2-O-α-D-glucopyranosyl-L-ascorbic acid according to claim 5, characterized in that, The final concentration of the substrate sucrose is 1.2 M to 1.4 M; the final concentration of the substrate vitamin C is 1.1 M to 1.3 M.

8. The preparation method of 2-O-α-D-glucopyranosyl-L-ascorbic acid according to claim 5, characterized in that, The dosage of the catalyst is 40 g / L based on the total weight of the immobilized cells; the final concentration of the substrate sucrose is 1.3 M; the final concentration of the substrate vitamin C is 1.2 M.

9. The preparation method of 2-O-α-D-glucopyranosyl-L-ascorbic acid according to claim 5, characterized in that, Reaction conditions: React at 40 °C, 300 rpm in the dark for 15 h.

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