Spytag / SpyCatcher cyclization modification-based sucrose phosphorylase mutant and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a sucrose phosphorylase mutant based on Spytag / SpyCatcher cyclization modification and application of the sucrose phosphorylase mutant. According to the present invention, the 257th-site serine and the 413th-site isoleucine of the wild-type sucrose phosphorylase of the sucrose phosphorylase (EfSPase) derived from enterococcus faecium are subjected to multi-site simultaneous mutation to obtain the mutation intermediate, and the Spytag and the SpyCatcher tag are respectively connected to the N end and the C end of the mutation intermediate to perform cyclization so as to obtain the sucrose phosphorylase mutant, wherein the Spytag and the SpyCatcher tag are respectively connected to the N end and the C end of the mutation intermediate; according to the sucrose phosphorylase mutant based on Spytag / SpyCatcher cyclization modification, the half-life period is prolonged to 3 times of that of a wild type at 45 DEG C (prolonged to 30 hours from 10 hours), the catalytic efficiency is greatly improved, the yield of 2-alpha-glycerol glucoside reaches 240.6 g / L, and the activity is kept 90% or above after the immobilized cyclase is continuously used 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 increase the understanding of the overall background 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] 2-α-glycosylglucose is ubiquitous 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-glycosylglucose 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-glycosylglucose is also used in the preparation of oral care products, such as oral sprays, toothpastes, etc., 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; therefore, 2-α-glycosylglucose has great application value and market prospects.

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

[0005] However, the currently reported sucrose phosphorylase and its mutants 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 objectives, the present invention adopts the following technical solutions: 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-terminal and C-terminal of the mutant intermediate respectively and cyclizing them.

[0008] 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.

[0009] In one or more embodiments, 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.

[0010] Preferably, the nucleotide sequence of linker1 is as shown in SEQ ID NO.9; The nucleotide sequence of linker2 is as shown in SEQ ID NO.10.

[0011] 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.

[0012] 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.

[0013] In the third aspect of the present invention, there is provided an expression cassette comprising the gene described in the second aspect.

[0014] In the fourth aspect of the present invention, there is provided a recombinant expression vector comprising the gene described in the second aspect.

[0015] In the fifth aspect of the present invention, there is provided a recombinant bacterium comprising the gene described in the second aspect.

[0016] In the sixth aspect of the present invention, there is provided a transgenic cell line comprising the gene described in the second aspect.

[0017] 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.

[0018] The eighth aspect of the present invention provides a method for synthesizing 2-α-glucosylglycerol, comprising: Using the wet cells obtained by induced cultivation 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; 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.

[0019] 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.

[0020] The beneficial effects of the present invention are as follows: 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 simultaneous multi-site mutation of 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 a 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1 ForE.coli Reaction formula for the catalytic synthesis of glucosylglycerol by BL21(DE3) / pET28a-Spytag-EfSPase-S257L-I413V-SpyCatcher; Figure 2 It is the map of the recombinant plasmid pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher. Specific implementation manners

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The culture medium formulations used in the following examples are as follows: 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.

[0027] 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.

[0028] 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 detector was used, the detection time was 20 min, and the flow rate was 1 mL / min. The column temperature was 40 °C.

[0029] 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.

[0030] Example 1 Construction of expression vector and engineered bacteria: Through the mining of gene libraries, a sucrose phosphorylase (EfSPase) derived from Enterococcus ( Enterococcus faecium ) was screened, with the NCBI accession number WP_002290364.1. It 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.

[0031] 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).

[0032] F1: 5’-ctttaagaaggagatataccATGAAAATCAAAAACGAAGCCAT-3’; R1: 5’- tggtggtggtggtgctcgagTTAAAACTTCAGGGCCACGG-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-EfSPase was constructed.

[0033] Construction of the expression plasmid: Under the priming 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-EfSPase.

[0034] Preparation of competent cells: Obtained from the glycerol tube stored in the -80 °C refrigerator E. coliThe BL21(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 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 medium, and cultured at 37 °C and 180 rpm until the OD 600 reached 0.4 - 0.6; the bacterial solution was pre-cooled on ice, taken into a sterilized centrifuge tube, placed on ice for 10 min, and centrifuged at 4 °C and 5000 rpm for 10 min; the supernatant was poured out, taking care to prevent contamination, and the precipitated cells were resuspended with pre-cooled 0.1 mol / L aqueous CaCl2 solution and placed on ice for 30 min; centrifuged at 4 °C and 5000 rpm for 10 min, the supernatant was discarded, and the precipitated cells were resuspended with pre-cooled 0.1 mol / L aqueous CaCl2 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.

[0035] 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 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. Clones were randomly picked and the plasmids were extracted for sequencing identification to screen and obtain recombinant Escherichia coli containing the recombinant plasmid for expression E.coli BL21(DE3) / pET28a-EfSPase.

[0036] Example 2 Induced expression of sucrose phosphorylase: 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 an 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 OD of the bacterial cells 600Reaching 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, 8000 rpm for 20 min, discard the supernatant, collect the precipitate, and thus obtain the wet cells of the recombinant strain pET28a - EfSPase containing sucrose phosphorylase.

[0037] Example 3 Establishment of the sucrose phosphorylase gene mutation library: Using the E.coli BL21(DE3) / pET28a - EfSPase constructed in Example 2 as the starting strain.

[0038] Modify it through the method of directed evolution theory. According to the calculation of protein folding free energy, select the sites G21K / S257L / I413V / A438E / I470T / K474N with improved stability for site - directed mutagenesis.

[0039] The mutation PCR system (100 μL) is 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 add ddH2O to make up to 50 μL. 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 results are respectively verified by positive DNA agarose gel electrophoresis. Digest the PCR product with DpnI enzyme for the template at 37 °C for 1 hour, 200 rpm, inactivate at 65 °C for 1 minute, transform the PCR product by heat shock, and activate Escherichia coli E. coli BL21(DE3), place it at 37 °C, 200 rpm, culture for 1 hour, spread it on an LB plate containing 50 μg / mL kanamycin resistance, and culture it inverted at 37 °C overnight.

[0040] Table 1 Design of primers for site - directed mutagenesis of sucrose phosphorylase

[0041] After DNA sequencing, the DNA sequencing results of the mutants with site - directed mutagenesis of G21K, S257L, I413V, A438E, I470T, K474N, and S257L + I413V are completely consistent with the expected design mutations.

[0042] Example 4 Screening of the sucrose phosphorylase gene mutation library: 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 OD 600 of the bacteria 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 bacteria containing the sucrose phosphorylase gene mutation library.

[0043] 1. Primary screening: Prepare the reaction solution (200 μL): Substrate sucrose with a final concentration of 100 mM, glycerol with a final concentration of 110 mM, and the catalyst dosage is 5 g / L based on the total weight of the wet bacteria. 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 ends, take 20 μL of the reaction sample, 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.

[0044] Table 2 Primary screening reaction results

[0045] 2. Secondary screening: Perform secondary screening on the strains obtained from the primary screening. Prepare the reaction solution (10 mL) for secondary screening: Substrate sucrose with a final concentration of 300 mM, glycerol with a final concentration of 330 mM, and the catalyst dosage is 5 g / L based on the total weight of the wet bacteria. 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 ends, take 20 μL of the reaction sample, dilute it 10 times, filter it through a 0.22 μm filter membrane, and perform HPLC detection. The detection results are shown in Table 3.

[0046] Table 3 Secondary screening reaction results

[0047] Example 5 Construction of sucrose phosphorylase mutants based on Spytag / SpyCatcher cyclization modification By connecting the sucrose phosphorylase mutant EfSPase-S257L-I413V with the highest activity obtained in Example 4 to SpyTag and SpyCatcher tags at the N-terminus and C-terminus of EfSPase-S257L-I413V, specifically: the N-terminus is connected to the Spytag tag through linker1; the C-terminus is 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 entrusted Nanjing Genscript Biotech Co., Ltd. to perform 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.

[0048] Place the crude enzyme solutions of EfSPase, EfSPase-S257L-I413V, and SpyTag-EfSPase-S257L-I413V-SpyCatcher in a 45 °C water bath for heat treatment. Samples are taken at different times to measure the enzyme activity. The enzyme activity reaction system (200 μL): the final concentration of the substrate sucrose is 100 mM, the final concentration of glycerol is 110 mM, the catalyst dosage is 5 g / L based on the total weight of the wet bacteria before disruption, and the reaction solution is composed of pure water as the reaction medium. 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. Calculate the half-life (t 1 / 2 ) of the enzyme by fitting the curve. The results are shown in Table 4.

[0049] Table 4 Results of the half-life determination of the enzyme

[0050] Example 6 Application of sucrose phosphorylase in the catalytic synthesis of glycerol glucoside: The recombinant sucrose phosphorylase mutant E.coliBL21(DE3) / pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher was inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg / mL and cultured at 37 °C for 9 hours to obtain a seed culture. Then, the seed culture was inoculated into a 5-L fermenter containing 3 L of fermentation medium at an inoculation volume of 3.5% (v / v). The prepared medium was added, and the air outlet and inlet were sealed tightly. After installation and sealing, the inoculation port was opened, and it was sterilized together with the prepared lactose inducer in an autoclave at 115 °C for 30 min. After sterilization, the fermenter was screwed with the inoculation port and installed on the operating system. Condensed water and air were introduced (a sterilizing membrane should be installed on the inlet pipe), and the air outlet was inserted below the liquid level in the conical flask. When the autoclave cooled down to 37 °C, a heating ring was placed around the inoculation port, and the cultured seed culture was inoculated into the fermenter. It was cultured at 37 °C and 500 rpm for about 3 - 4 h until the cell density OD 600 reached 6 - 8. After the temperature of the fermenter was lowered to 25 °C, lactose at a final concentration of 16 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 wet cells containing the sucrose phosphorylase mutant E.coli BL21(DE3) / pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher.

[0051] 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. Distilled water was added to make the volume up to 3 L and dissolved.

[0052] The amount of catalyst was 15 g / L based on the total weight of the wet cells. The final concentration of the substrate sucrose was 1 M, the final concentration of the substrate glycerol was 1.1 M, and pure water was used as the reaction medium to make the total volume of the reaction solution 1 L. Reaction conditions: 45 °C, 500 rpm for 12 h. After the reaction, 20 μL of the reaction sample was taken, diluted 50 times, filtered through a 0.22-μm filter membrane, and detected by HPLC. The concentration of 2-α-glucosylglycerol after the reaction was 240.6 g / L (0.94 M).

[0053] Example 7 Immobilization of cyclase SpyTag-EfSPase-S257L-I413V-SpyCatcher and its application in the catalytic synthesis of 2-glycosylglycerol Take E.coliThe crude enzyme solution of BL21(DE3) / pET28a-SpyTag-EfSPase-S257L-I413V-SpyCatcher wet bacteria was obtained by ultrasonic disruption. An appropriate amount of diatomaceous earth was weighed and added (the mass ratio of diatomaceous earth to wet bacteria was 5:1), stirred and mixed evenly for 10 - 15 min, then flocculated with 0.15% (by volume) of polyethyleneimine for 20 - 30 min, and finally covalently cross-linked with 0.5% (by volume) of glutaraldehyde for 1 h, and then washed 3 - 5 times with pure water to obtain the immobilized SpyTag-EfSPase-S257L-I413V-SpyCatcher.

[0054] The catalyst dosage was 25 g / L based on the total weight of the immobilized enzyme, the final concentration of the substrate sucrose was 1 M, the final concentration of the substrate glycerol was 1.1 M, and pure water was used as the reaction medium to make the total volume of the reaction solution 1 L. The reaction conditions were: 45 °C, 500 rpm for 12 h. After the reaction ended, 20 μL of the reaction sample was taken, diluted 50 times, filtered through a 0.22 μm filter membrane, and detected by HPLC. The concentration of 2-α-glyceroglucoside after the immobilized SpyTag-EfSPase-S257L-I413V-SpyCatcher reacted for 12 h was 236.8 g / L (0.93 M), and after reacting for 10 batches, the enzyme activity could still be maintained above 90%.

[0055] 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 changes and modifications. 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 simultaneously mutating multiple sites of serine at position 257 and isoleucine at position 413 of the wild-type sucrose phosphorylase with an amino acid sequence as shown in SEQ ID NO.2 to obtain a mutant intermediate, and then cyclizing by connecting Spytag and SpyCatcher tags to the N-terminus and C-terminus of the mutant intermediate respectively.

2. The sucrose phosphorylase mutant according to claim 1, wherein 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; 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; the nucleotide sequence of linker1 is as shown in SEQ ID NO.9; the nucleotide sequence of linker2 is as shown in SEQ ID NO.10; The mutant intermediate of the sucrose phosphorylase is obtained by mutating serine at position 257 of the wild-type sucrose phosphorylase with an amino acid sequence as shown in SEQ ID NO.2 to leucine and mutating isoleucine at position 413 to valine.

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, the gene according to claim 3, or the recombinant bacterium according to claim 6 in the catalytic synthesis of 2-α-glycosylglucose.

9. A method for synthesizing 2-α-glucosylglycerol, characterized in that, Including: Using the wet cells obtained by induced culture of a 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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