A sucrose phosphorylase mutant based on Spytag / SpyCatcher cyclization modification and its application

Abstract

The present invention relates to the field of bioengineering technology, and specifically relates to a sucrose phosphorylase mutant based on Spytag / SpyCatcher cyclization modification and its application. In the present invention, a mutant intermediate is obtained by simultaneously mutating multiple sites of serine at position 257 and isoleucine at position 413 of the wild-type sucrose phosphorylase derived from Enterococcus, and is obtained after cyclization by connecting Spytag and SpyCatcher tags to the N-terminus and C-terminus of the mutant intermediate respectively. The half-life of the mutant at 45 °C is extended to 3 times that of the wild-type, and the catalytic efficiency is greatly improved. The yield of 2-α-glycosylglucose reaches 240.6 g / L, and the activity of the immobilized cyclase remains above 90% after continuous use for 10 batches.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a sucrose phosphorylase mutant based on Spytag / SpyCatcher cyclization modification and its application. 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 indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] 2-α-Glucosylglycerol exists widely in nature, especially in salt-tolerant cyanobacteria such as blue-green algae and Myrothamnus flabellifolius Welw. (also known as resurrection plant). It is the most important active substance for Myrothamnus flabellifolius Welw. to survive and reactivate in extreme environments, and can firmly lock the precious last drop of water in the body. Therefore, 2-α-glucosylglycerol has extremely strong physiological effects of "moisturizing, water-locking, and moisturizing", and can be used as a functional raw material for cosmetics; in addition, 2-α-glucosylglycerol is also used in the preparation of oral care products such as oral sprays and toothpastes, which have anti-inflammatory, antibacterial, and analgesic effects; in the food field, it can be used in the preparation of foods such as beverages and candies to improve the quality and shelf life of foods; thus, 2-α-glucosylglycerol has great application value and market prospects.

[0004] At present, the preparation of 2-α-glucosylglycerol mainly uses the biocatalytic method for synthesis. Plant extraction is limited by site, source, etc. and cannot be produced on a large scale; chemical synthesis has problems of more impurities (1-glucosylglycerol will be introduced during synthesis, and 2-α-glucosylglycerol has the best water-locking and moisturizing functions), so the biocatalytic method with high specificity is mainly used for synthesis at present. Using sucrose and glycerol as raw materials, 2-glucosylglycerol is produced under the action of sucrose phosphorylase.

[0005] However, the reported sucrose phosphorylase and its mutants currently have problems of low enzyme activity and insufficient stability, so they do not meet the requirements of industrial production. Summary of the Invention

[0006] In order to overcome the above problems, the present invention provides a sucrose phosphorylase mutant based on Spytag / SpyCatcher cyclization modification and its application.

[0007] To achieve the above technical purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, there is provided a sucrose phosphorylase mutant modified by Spytag / SpyCatcher cyclization, which is obtained by simultaneously mutating multiple sites of serine at position 257 and isoleucine at position 413 of the wild-type sucrose phosphorylase with the amino acid sequence shown in SEQ ID NO.2 to obtain a mutant intermediate, and then connecting Spytag and SpyCatcher tags to the N-terminus and C-terminus of the mutant intermediate respectively and cyclizing them.

[0009] In one or more embodiments, the nucleotide sequence of the Spytag tag is as shown in SEQ ID NO.7, and the nucleotide sequence of the SpyCatcher tag is as shown in SEQ ID NO.8.

[0010] In one or more embodiments, the N-terminus of the mutant intermediate is connected to the Spytag tag through linker1; the C-terminus of the mutant intermediate is connected to the SpyCatcher tag through linker2.

[0011] Preferably, the nucleotide sequence of linker1 is as shown in SEQ ID NO.9;

[0012] The nucleotide sequence of linker2 is as shown in SEQ ID NO.10.

[0013] In one or more embodiments, the sucrose phosphorylase mutant intermediate is obtained by mutating serine at position 257 of the wild-type sucrose phosphorylase with the amino acid sequence shown in SEQID NO.2 to leucine, and mutating isoleucine at position 413 to valine.

[0014] In the second aspect of the present invention, there is provided a gene encoding the sucrose phosphorylase mutant modified by Spytag / SpyCatcher cyclization described in the first aspect.

[0015] In the third aspect of the present invention, there is provided an expression cassette comprising the gene described in the second aspect.

[0016] In the fourth aspect of the present invention, there is provided a recombinant expression vector comprising the gene described in the second aspect.

[0017] In the fifth aspect of the present invention, there is provided a recombinant bacterium comprising the gene described in the second aspect.

[0018] In the sixth aspect of the present invention, there is provided a transgenic cell line comprising the gene described in the second aspect.

[0019] The seventh aspect of the present invention provides the use of the sucrose phosphorylase mutant modified by Spytag / SpyCatcher cyclization 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-α-glucosylglycerol.

[0020] The eighth aspect of the present invention provides a method for synthesizing 2-α-glucosylglycerol, comprising:

[0021] Using the wet cells obtained by induced culture of a genetically engineered bacterium of a sucrose phosphorylase mutant modified by Spytag / SpyCatcher cyclization, or the crude enzyme solution extracted by ultrasonic disruption of the wet cells, or the immobilized enzyme as a catalyst, using sucrose and glycerol as substrates, and pure water as a reaction medium to form a reaction system, and reacting to obtain 2-α-glucosylglycerol;

[0022] Wherein, the genetically engineered bacterium is constructed by introducing the sucrose phosphorylase mutant modified by Spytag / SpyCatcher cyclization described in the first aspect into a host bacterium.

[0023] In one or more embodiments, the dosage of the catalyst is 5-15 g / L based on the total weight of the wet cells, the final concentration of the substrate sucrose is 0.9-1.2 M, preferably 1 M; the final concentration of the substrate glycerol is 1.0-1.3 M, preferably 1.1 M.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention relates to the field of bioengineering technology, and specifically relates to a sucrose phosphorylase mutant modified by Spytag / SpyCatcher cyclization and its application. In the present invention, a mutant intermediate is obtained by simultaneously mutating serine at position 257 and isoleucine at position 413 of the wild-type sucrose phosphorylase of sucrose phosphorylase (EfSPase) derived from Enterococcus ( Enterococcus faecium ). After connecting Spytag and SpyCatcher tags to the N-terminus and C-terminus of the mutant intermediate respectively and cyclizing, the sucrose phosphorylase mutant modified by Spytag / SpyCatcher cyclization has its half-life at 45 °C extended to 3 times that of the wild-type (from 10 hours to 30 hours), and the catalytic efficiency has been greatly improved. The yield of 2-α-glucosylglycerol reaches 240.6 g / L, and the activity of the immobilized cyclized enzyme remains above 90% after continuous use for 10 batches. Description of the Drawings

[0026] The specification drawings constituting 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 of the present invention.

[0027] Figure 1 For E.coli Reaction formula for the catalytic synthesis of glucosylglycerol by BL21(DE3) / pET28a-Spytag-EfSPase-S257L-I413V-SpyCatcher;

[0028] Figure 2 It is the map of the recombinant plasmid pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher. Detailed implementation manners

[0029] 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.

[0030] 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 form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0031] 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 combination with specific embodiments.

[0032] The medium formulations used in the following examples are as follows:

[0033] LB 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.

[0034] 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.

[0035] The concentration of the product glucosylglycerol was detected by high performance liquid chromatography (HPLC), and the analysis method was as follows: Column model: Waters XBridge Amide (4.6 mm × 250 mm, 5 μm). The mobile phase was A (water):B (acetonitrile) = 15:85, the injection volume was 10 μL, a differential refractive index detector was used, the detection time was 20 min, the flow rate was 1 mL / min. The column temperature was 40 °C.

[0036] Sample treatment: Take 20 μL of the sample after the reaction, dilute it 30 times with 85% acetonitrile solution, filter it through a 0.22 μm filter membrane, and perform HPLC detection.

[0037] Example 1

[0038] Construction of expression vector and engineered bacteria:

[0039] Through the mining of the gene library, a sucrose phosphorylase (EfSPase) derived from Enterococcus ( Enterococcus faecium ) was screened, with the NCBI accession number WP_002290364.1. It was commissioned 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.

[0040] According to the nucleotide sequence shown in SEQ ID NO.1 and the pET-28a vector sequence, primers F1, R1, F2, and R2 were designed (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).

[0041] F1: 5’-ctttaagaaggagatataccATGAAAATCAAAAACGAAGCCAT-3’;

[0042] R1: 5’- tggtggtggtggtgctcgagTTAAAACTTCAGGGCCACGG-3’;

[0043] F2: 5’-CTCGAGCACCACCACCACC-3’;

[0044] R2: 5’-GGTATATCTCCTTCTTAAAGTTAAACAAAAT-3’;

[0045] Using the pET-28a plasmid as the expression vector, Escherichia coli E.coli BL21(DE3) / pET-28a-EfSPase was constructed.

[0046] Construction of 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 by using high-fidelity Pfu DNA polymerase. Using the pET-28a plasmid as a template, the linearized vector sequence was amplified by using high-fidelity Pfu DNA polymerase, and the target gene was homologously recombined with the linearized vector by using homologous recombinase to construct plasmid pET28a-EfSPase.

[0047] Preparation of competent cells: Obtain the glycerol stock-preserved 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 the single colonies on the LB plate and inoculate them into a test tube containing 5 mL of LB medium, and culture them 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 medium, and culture it at 37 °C and 180 rpm until the OD 600 reaches 0.4 - 0.6; pre-cool the bacterial solution on ice, transfer the bacterial solution to 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, pay attention to preventing 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, and store them in the -80 °C refrigerator and take them out when needed.

[0048] Construction of recombinant Escherichia coli: First, place 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 medium, and culture it on a shaker at 37 °C and 200 rpm for 1 h; spread it on an LB plate containing 50 μg / mL kanamycin resistance and culture it at 37 °C for 8 - 12 h. Randomly pick clones to extract plasmids for sequencing identification, and screen to obtain recombinant Escherichia coli E.coli BL21(DE3) / pET28a-EfSPase.

[0049] Example 2

[0050] Induced expression of sucrose phosphorylase:

[0051] Wet cells containing the sucrose phosphorylase gene: The recombinant Escherichia coli obtained in Example 1 was respectively E.coli BL21(DE3) / pET28a-EfSPase 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 cell OD 600 reached 0.6 - 0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM was added, and after inducing culture at 25 °C for 16 h, centrifuged at 4 °C and 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected to obtain the wet cells of the recombinant strain pET28a-EfSPase containing sucrose phosphorylase.

[0052] Example 3

[0053] Establishment of the sucrose phosphorylase gene mutation library:

[0054] Using the E.coli BL21(DE3) / pET28a-EfSPase constructed in Example 2 as the starting strain.

[0055] It was modified by the method of directed evolution theory. According to the calculation of protein folding free energy, the sites G21K / S257L / I413V / A438E / I470T / K474N with improved stability were selected for site-directed mutagenesis.

[0056] The mutation PCR system (100 μL) was: 25 μL of 2×Phanta Max buffer, 1 μL of dNTPs, 1 μL of each of the mutation upstream and downstream primers, 1 μL of the template (starting strain), 0.5 μL of Pfu DNA polymerase, and ddH2O was added 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 results were respectively verified by DNA agarose gel electrophoresis for positivity. The PCR products were digested with DpnI enzyme for the template, incubated at 37 °C for 1 hour and 200 rpm, inactivated at 65 °C for 1 minute, the PCR products were heat-shock transformed, and the Escherichia coli E. coli BL21(DE3) was activated, cultured at 37 °C and 200 rpm for 1 hour, spread on an LB plate containing 50 μg / mL kanamycin resistance, and incubated upside down at 37 °C overnight.

[0057] Table 1 Design of Primers for Site-directed Mutagenesis of Sucrose Phosphorylase

[0058]

[0059] After DNA sequencing, the DNA sequencing results of the mutants with site-directed mutations of G21K, S257L, I413V, A438E, I470T, K474N, and S257L+I413V were completely consistent with the expected designed mutations.

[0060] Example 4

[0061] Screening of the Gene Mutation Library of Sucrose Phosphorylase:

[0062] Pick monoclonal colonies from the 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, 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, 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 cells containing the gene mutation library of sucrose phosphorylase.

[0063] 1. Primary Screening:

[0064] Prepare the reaction solution (200 μL): sucrose with a final concentration of 100 mM as the substrate, glycerol with a final concentration of 110 mM, and the catalyst dosage is 5 g / L based on the total weight of the wet cells. Use pure water as the reaction medium to form the reaction solution. Reaction conditions: React on a reactor at 45 °C and 500 rpm for 2 h. After the reaction, take 20 μL of the sample at the end of the reaction, dilute it 5 times, filter it through a 0.22 μm filter membrane, and perform HPLC detection. The detection results are shown in Table 2.

[0065] Table 2 Results of Primary Screening Reactions

[0066]

[0067] 2. Secondary Screening:

[0068] The strains obtained from the primary screening were subjected to secondary screening. The reaction solution (10 mL) for secondary screening was prepared as follows: the final concentration of the substrate sucrose was 300 mM, the final concentration of glycerol was 330 mM, the amount of the catalyst was 5 g / L based on the total weight of the wet cells, and pure water was used as the reaction medium to form the reaction solution. Reaction conditions: The reaction was carried out on a reactor at 45 °C and 500 rpm for 2 hours. After the reaction ended, 20 μL of the sample at the end of the reaction was taken, diluted 10 times, filtered through a 0.22 μm filter membrane, and subjected to HPLC detection. The detection results are shown in Table 3.

[0069] Table 3 Results of the secondary screening reaction

[0070]

[0071] Example 5

[0072] Construction of sucrose phosphorylase mutants based on Spytag / SpyCatcher cyclization modification

[0073] By connecting the SpyTag and SpyCatcher tags to the N-terminus and C-terminus of the sucrose phosphorylase mutant EfSPase-S257L-I413V with the highest activity obtained in Example 4, specifically: the N-terminus was connected to the Spytag tag through linker1; the C-terminus was connected to the SpyCatcher tag through linker2 to construct the cyclase SpyTag-EfSPase-S257L-I413V-SpyCatcher (see the plasmid map for construction in Figure 2 ), and Nanjing Genscript Biotech Co., Ltd. was commissioned for total gene synthesis to obtain the recombinant plasmid pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher. The specific transformation and induction expression process of the strain E.coli BL21(DE3) / pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher refer to Examples 1 and 2.

[0074] The crude enzyme solutions of EfSPase, EfSPase-S257L-I413V, and SpyTag-EfSPase-S257L-I413V-SpyCatcher were subjected to heat treatment in a 45 °C water bath. Samples were taken at different times to measure the enzyme activity. The enzyme activity reaction system (200 μL): sucrose with a final concentration of 100 mM as the substrate, glycerol with a final concentration of 110 mM, and the catalyst dosage was 5 g / L based on the total weight of wet bacterial cells before disruption. The reaction medium was pure water to form the reaction solution. Reaction conditions: React on a reactor at 45 °C and 500 rpm for 2 hours. After the reaction, take 20 μL of the sample at the end of the reaction, dilute it 5 times, filter it through a 0.22 μm filter membrane, and perform HPLC detection. By fitting the curve, calculate the half-life (t 1 / 2 ). The results are shown in Table 4.

[0075] Table 4 Results of the determination of the half-life of the enzyme

[0076]

[0077] Example 6

[0078] Application of sucrose phosphorylase in the catalytic synthesis of glycerol glucoside:

[0079] The recombinant sucrose phosphorylase mutant E.coli BL21(DE3) / pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher was inoculated into LB liquid medium containing kanamycin with a final concentration of 50 μg / mL and cultured at 37 °C for 9 hours as the seed liquid. It was then inoculated into a 5 L fermenter containing 3 L of fermentation medium at an inoculation volume concentration of 3.5%. The prepared medium was added, the air outlet and inlet were sealed, installed and sealed well, the inoculation port was opened, and it was placed in an autoclave with the prepared lactose inducer for sterilization at 115 °C for 30 min. The sterilized fermenter was screwed with the inoculation port and installed on the operating system, and condensed water and air were passed through (a sterilization membrane should be installed on the inlet pipe). The air outlet was inserted below the liquid level of the conical flask. When the autoclave cooled to 37 °C, a heating ring was placed on the inoculation port, and the cultured seed liquid was inoculated into the fermenter. It was cultured at 37 °C and 500 rpm for about 3 - 4 h. When the cell density OD 600 reached 6 - 8, which met the requirements. After the temperature of the fermenter was lowered to 25 °C, lactose with a final concentration of 16 g / L was added as the 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-SpyTag-EfSPase-S257L-I413V-SpyCatcher.

[0080] The composition of the fermenter medium: 45 g of tryptone, 36 g of yeast extract, 30 g of sodium chloride, 4.08 g of potassium dihydrogen phosphate, 45 g of glycerol, 6.84 g of dipotassium hydrogen phosphate trihydrate, 15 g of ammonium sulfate, 1.125 g of magnesium sulfate, 4 g of antifoaming agent. Add distilled water to make the volume up to 3 L and dissolve.

[0081] The dosage of the catalyst is 15 g / L based on the total weight of the wet cells. The final concentration of the substrate sucrose is 1 M, and the final concentration of the substrate glycerol is 1.1 M. Pure water is used as the reaction medium, and the total volume of the reaction solution is 1 L. Reaction conditions: React at 45 °C and 500 rpm for 12 hours. After the reaction, take 20 μL of the sample at the end of the reaction, dilute it 50 times, filter it through a 0.22 μm filter membrane, and perform HPLC detection. The concentration of 2-α-glucosylglycerol at the end of the reaction is 240.6 g / L (0.94 M).

[0082] Example 7

[0083] Immobilization of cyclase SpyTag-EfSPase-S257L-I413V-SpyCatcher and its application in the catalytic synthesis of 2-glucosylglycerol

[0084] Take E.coli The wet cells of BL21(DE3) / pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher are ultrasonically disrupted to obtain a crude enzyme solution. Weigh an appropriate amount of diatomaceous earth and add it (the mass ratio of diatomaceous earth to wet cells is 5:1), stir and mix evenly for 10 - 15 min, then add 0.15% (v / v) polyethyleneimine to flocculate for 20 - 30 min, and finally add 0.5% (v / v) glutaraldehyde for covalent crosslinking for 1 h. Then wash it 3 - 5 times with pure water to obtain the immobilized SpyTag-EfSPase-S257L-I413V-SpyCatcher.

[0085] The amount of catalyst is 25 g / L based on the total weight of the immobilized enzyme, the final concentration of the substrate sucrose is 1 M, the final concentration of the substrate glycerol is 1.1 M, and pure water is used as the reaction medium to make the total volume of the reaction solution 1 L. Reaction conditions: react at 45 °C and 500 rpm for 12 hours. After the reaction, take 20 μL of the sample at the end of the reaction, dilute it 50 times, filter it through a 0.22 μm filter membrane, and perform HPLC detection. After the immobilized SpyTag-EfSPase-S257L-I413V-SpyCatcher reacts for 12 h, the concentration of 2-α-glycosylglucose is 236.8 g / L (0.93 M), and after reacting for 10 batches, the enzyme activity can still remain above 90%.

[0086] The above are only the preferred embodiments of the present invention and are not intended 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 based on Spytag / SpyCatcher cyclization modification, characterized in that, It is obtained by mutating serine at position 257 and isoleucine at position 413 of the wild-type sucrose phosphorylase with the amino acid sequence shown in SEQ ID NO.2 to leucine and valine respectively to obtain a mutant intermediate, and then cyclizing by connecting Spytag and SpyCatcher tags to the N-terminal and C-terminal of the mutant intermediate respectively.

2. The sucrose phosphorylase mutant according to claim 1, characterized in that, The N-terminal of the mutant intermediate is connected to the Spytag tag through linker1; the C-terminal of the mutant intermediate is connected to the SpyCatcher tag through linker2; the nucleotide sequence of linker1 is shown in SEQ ID NO.9; the nucleotide sequence of linker2 is shown in SEQ ID NO.10; The nucleotide sequence of the Spytag tag is shown in SEQ ID NO.7, and the nucleotide sequence of the SpyCatcher tag is shown in SEQ ID NO.

8.

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

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

5. A recombinant expression vector, characterized in that, Comprising the gene according to claim 3.

6. A recombinant bacterium, characterized in that, Comprising the gene according to claim 3.

7. A transgenic cell line, characterized in that, Comprising the gene according to claim 3.

8. Use of the sucrose phosphorylase mutant according to claim 1 or 2 or the recombinant bacterium according to claim 6 in the catalytic synthesis of 2-α-glycosylglucose.

9. A method for synthesizing 2-α-glycosylglucose, characterized in that, Including: Using the wet cells obtained by induced culture of the genetically engineered bacterium based on the Spytag / SpyCatcher cyclized modified 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 glycerol as substrates, and pure water as a reaction medium to form a reaction system, and reacting to obtain 2-α-glycosylglucose; Wherein, the genetically engineered bacterium is constructed by introducing the sucrose phosphorylase mutant according to claim 1 or 2 into a host bacterium.

10. The method according to claim 9, wherein The dosage of the catalyst is 5-15 g / L based on the total weight of the wet cells, the final concentration of the substrate sucrose is 0.9-1.2 M, and the final concentration of the substrate glycerol is 1.0-1.3 M.

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