Mutant sucrose phosphorylase and application thereof in catalytic synthesis of L-ascorbic acid 2-O-alpha-D-glucoside

Abstract

The invention provides mutant sucrose phosphorylase and application of the mutant sucrose phosphorylase in catalytic synthesis of L-ascorbic acid 2-O-alpha-D-glucoside, and belongs to the technical field of enzyme catalyst preparation. The mutant sucrose phosphorylase is obtained by point mutation of wild sucrose phosphorylase; the amino acid sequence of the wild type sucrose phosphorylase is as shown in SEQ ID NO. 1; the mutant type sucrose phosphorylase comprises one or more of the following site mutations: A84G, G147L, V233L, Y237F, K238D and N402A. The mutant type sucrose phosphorylase is a mutant type sucrose phosphorylase. Compared with wild type sucrose phosphorylase, the multiple mutant sucrose phosphorylase provided by the invention has remarkable advantages in the aspect of catalytic activity. The mutant sucrose phosphorylase provided by the invention can realize high-yield and large-scale industrial production of L-ascorbic acid 2-O-alpha-D-glucoside at different reaction temperatures and high substrate concentrations.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalyst preparation technology, and particularly relates to a mutant sucrose phosphorylase and its application in the catalytic synthesis of L-ascorbic acid 2-O-α-D-glucoside. Background Technology

[0002] L-Ascorbic acid 2-O-α-D-glucoside (AA-2G) is a vitamin C derivative with excellent stability. It is prepared by binding a glucose molecule to the 2-hydroxyl group of L-ascorbic acid (vitamin C). Its core function is to serve as a long-acting and stable source of vitamin C, and it is widely used in the food, cosmetics and pharmaceutical fields.

[0003] The preparation of L-ascorbic acid 2-O-α-D-glucoside (AA-2G) is mainly carried out by enzymatic synthesis, supplemented by chemical synthesis and microbial transformation. Among these methods, enzymatic catalysis is the preferred choice for industrial production due to its mild reaction conditions, high selectivity, and high product purity. Compared with chemical synthesis, enzymatic catalysis for preparing high-value glycosides has advantages such as high catalytic efficiency, simple process flow, and ease of industrial application. The enzymatic method for preparing L-ascorbic acid 2-O-α-D-glucoside involves cloning and expressing the sucrose phosphorylase gene in engineered bacteria, using sucrose and L-ascorbic acid as substrates, to establish a method for preparing L-ascorbic acid 2-O-α-D-glucoside based on sucrose phosphorylase. Currently, the highest yield of L-ascorbic acid 2-O-α-D-glucoside prepared by whole-cell catalysis in *E. coli* is 244 g / L (Enhancing regioselectivity of sucrose phosphorylase by loop engineering for glycosylation of L-ascorbic acid, *Applied Microbiology and Biotechnology*, 2022, 106, 4575-4586). This method involves... Bifidobacterium breve The mutant sucrose phosphorylase derived from this source was heterologously expressed in *E. coli*, and the L-ascorbic acid conversion rate was 64% after 72 h of whole-cell catalysis at 40°C. However, the reaction product of the above method for preparing L-ascorbic acid 2-O-α-D-glucoside contains 13-O-α-D-glucosyl-2-O-α-D-glucosyl-L-ascorbic acid and L-ascorbic acid 3-O-α-D-glucoside byproducts, which seriously affects the yield of the target product and makes the subsequent separation and purification process more complicated. Therefore, there is an urgent need for a sucrose phosphorylase with strong substrate selectivity, specific product configuration, and high catalytic activity to meet the needs of large-scale industrial production of L-ascorbic acid 2-O-α-D-glucoside. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a mutant sucrose phosphorylase and its application in the catalytic synthesis of L-ascorbic acid 2-O-α-D-glucoside. This invention provides a variety of mutant sucrose phosphorylases with different optimal reaction temperatures, high catalytic activity, and strong substrate specificity, enabling high-yield and large-scale industrial production of L-ascorbic acid 2-O-α-D-glucoside at different reaction temperatures and high substrate concentrations.

[0005] This invention provides a mutant sucrose phosphorylase, which is obtained by point mutation of wild-type sucrose phosphorylase. The amino acid sequence of the wild-type sucrose phosphorylase is shown in SEQ ID NO.1. The mutant sucrose phosphorylase contains one or more of the following site mutations: A84G, G147L, V233L, Y237F, K238D, and N402A.

[0006] Preferably, the mutant sucrose phosphorylase comprises one of the following two-site combined mutations: A84G / V233L, A84G / G147L, A84G / N402A, V233L / Y237F, V233L / K238D, V233L / N402A, Y237F / N402A, or K238D / N402A.

[0007] Preferably, the mutant sucrose phosphorylase comprises one of the following three-point combination mutations: A84G / V233L / Y237F, G147L / K238D / N402A, V233L / K238D / N402A, Y237F / K238D / N402A, G147L / Y237F / K238D.

[0008] Preferably, the mutant sucrose phosphorylase comprises one of the following four-site combined mutations: A84G / G147L / V233L / Y237F, A84G / G147L / V233L / K238D, G147L / V233L / K238D / N402A, V233L / Y237F / K238D / N402A.

[0009] Preferably, the mutant sucrose phosphorylase comprises one of the following five-site combination mutations: A84G / G147L / V233L / Y237F / K238D, A84G / G147L / V233L / Y237F / N402A, A84G / V233L / Y237F / K238D / N402A, G147L / V233L / Y237F / K238D / N402A.

[0010] Preferably, the mutant sucrose phosphorylase contains the following six-site combination mutations: A84G / G147L / V233L / Y237F / K238D / N402A.

[0011] This invention provides a DNA molecule encoding the mutant sucrose phosphorylase described above.

[0012] This invention provides the application of the mutant sucrose phosphorylase in the catalytic synthesis of L-ascorbic acid 2-O-α-D-glucoside.

[0013] Preferably, a reaction system is obtained by mixing sucrose, mutant sucrose phosphorylase and L-ascorbic acid, and L-ascorbic acid catalyzed reaction is carried out to prepare L-ascorbic acid 2-O-α-D-glucoside; The concentration of L-ascorbic acid in the reaction system is 0.2~2.6 mol / L, the mutant sucrose phosphorylase is used in the form of a whole-cell catalyst, and the concentration of mutant sucrose phosphorylase in the whole-cell catalyst is 0.5~6 mg / mL; the concentration of sucrose in the reaction system is 1~8 mol / L.

[0014] Preferably, the temperature of the enzyme-catalyzed reaction is 20℃~60℃, the time of the enzyme-catalyzed reaction is 12 h~72 h, the pH value of the enzyme-catalyzed reaction is 4~6, and the volume of the reaction system is 5~2000L.

[0015] Compared with existing technologies, the present invention has the following beneficial effects: The present invention obtains a variety of mutant sucrose phosphorylases by screening for beneficial mutation sites through saturation mutation, which have significant advantages over wild-type sucrose phosphorylases in terms of catalytic activity. The mutant sucrose phosphorylases provided by the present invention can achieve high-yield, large-scale industrial production of L-ascorbic acid 2-O-α-D-glucoside under different reaction temperatures and high substrate concentrations.

[0016] Furthermore, the mutant sucrose phosphorylase A84G / G147L / V233L / Y237F provided by this invention has an optimal reaction temperature as high as 40℃. In a 1000 L bioreactor, it can almost completely convert 1.4 M sucrose and 1.8 M L-ascorbic acid into L-ascorbic acid 2-O-α-D-glucoside within 48 h, with a maximum yield of 435 g / L. This mutant sucrose phosphorylase has high catalytic activity, mild reaction conditions, is green and low-carbon, and has high safety, showing promising prospects for industrialization. Attached Figure Description

[0017] Figure 1 The relative activities of wild-type sucrose phosphorylase and single-site mutant and double-site combined mutant sucrose phosphorylase at different temperatures; Figure 2 The relative activities of three-site, four-site, five-site, and six-site mutant sucrose phosphorylases at different temperatures were measured. Detailed Implementation

[0018] This invention provides a mutant sucrose phosphorylase, which is obtained by point mutation of wild-type sucrose phosphorylase. The amino acid sequence of the wild-type sucrose phosphorylase is shown in SEQ ID NO.1, and is as follows: MKNKVQLITYADRLGDGTLSSMADILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVD A IVNHMSWESKQFQDVLEKGEESEYYPMFLTMSSVFPNGATEEDLAGIYRPRPGLPFTHYKFA G KTRLVWVSFTPQQVDIDTSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIE V HSY YK KQVEIASKVDRVYDFALPPLLLHSLFTGHVEPVAHWTEIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNLDLYQVNSTYYSALGCNDQHYLAARAVQFFLPGVPQVYYVGALAGRNDMELLRRTNNGRDI N RHYYSTAEIDENLERPVVKALNALAKFRNELPAFDGEFSYEVDGDTSITFRWTAADGTSTAALTFEPGRGLGTDNATPVASLAWSDAAGDHETRDLLANPPIADID (where the bold underlined part indicates the mutation site).

[0019] In this invention, the mutant sucrose phosphorylase contains one or more of the following site mutations: A84G, G147L, V233L, Y237F, K238D, and N402A. This invention determines the mutation sites through sequence alignment and substrate tunneling analysis, uses the degenerate codon NNK to perform saturation mutations on multiple sites, screens for mutants with high activity, and thus obtains the aforementioned mutant sucrose phosphorylase.

[0020] Preferably, the mutant sucrose phosphorylase comprises one of the following two-site combined mutations: A84G / V233L, A84G / G147L, A84G / N402A, V233L / Y237F, V233L / K238D, V233L / N402A, Y237F / N402A, or K238D / N402A.

[0021] Preferably, the mutant sucrose phosphorylase comprises one of the following three-point combination mutations: A84G / V233L / Y237F, G147L / K238D / N402A, V233L / K238D / N402A, Y237F / K238D / N402A, G147L / Y237F / K238D.

[0022] Preferably, the mutant sucrose phosphorylase comprises one of the following four-site combined mutations: A84G / G147L / V233L / Y237F, A84G / G147L / V233L / K238D, A84G / G147L / Y237F / N402A, G147L / V233L / Y237F / K238D, G147L / V233L / K238D / N402A, V233L / Y237F / K238D / N402A.

[0023] Preferably, the mutant sucrose phosphorylase comprises one of the following five-site combination mutations: A84G / G147L / V233L / Y237F / K238D, A84G / G147L / V233L / Y237F / N402A, A84G / V233L / Y237F / K238D / N402A, G147L / V233L / Y237F / K238D / N402A.

[0024] Preferably, the mutant sucrose phosphorylase contains the following six-site combination mutations: A84G / G147L / V233L / Y237F / K238D / N402A.

[0025] In the specific implementation of this invention, the mutant sucrose phosphorylase A84G / G147L / V233L / Y237F exhibits the highest catalytic activity.

[0026] This invention provides a DNA molecule encoding the mutant sucrose phosphorylase described above. In this invention, the nucleotide sequence of the gene encoding the wild-type sucrose phosphorylase is shown in the NCBI database as AAO84039.1, specifically as follows (SEQ ID NO.2): ATGAAGAATAAAGTGCAGCTGATCACCTACGCCGATCGTCTGGGTGATGGTACCCTGAGCAGTATGGCAGATATTCTGCGTACCCGTTTTGATGGTGTTTATGATGGTGTTCATATCCTGCCGTTTTTCACCCCGTTTGATGGTGCAGATGCAGGTTTTGATCCGATTGATCATACCAAAGTTGACGAGCGTCTGGGCAGCTGGGATGATGTTGCAGAACTGAGCAAAACCCATAATATTATGGTGGAC GCC ATCGTGAATCACATGAGCTGGGAAAGCAAACAGTTTCAGGATGTTCTGGAAAAAGGCGAGGAAAGCGAATATTATCCGATGTTTCTGACCATGAGCAGCGTTTTTCCGAATGGTGCAACCGAAGAAGATCTGGCAGGTATTTATCGTCCGCGTCCGGGTCTGCCGTTTACCCATTATAAATTTGCA GGT AAAACCCGCCTGGTGTGGGTTAGCTTTACCCCGCAACAAGTTGATATTGATACCGATAGCGATAAAGGCTGGGAATATCTGATGAGCATTTTCGATCAGATGGCCGCAAGCCATGTTAGCTATATTCGTCTGGATGCAGTTGGTTATGGTGCAAAAGAAGCAGGTACCAGCTGTTTTATGACCCCGAAAACCTTTAAACTGATCAGCCGTCTGCGTGAAGAAGGTGTTAAACGTGGTCTGGAAATTCTGATTGAA GTG CATAGCTAT TACAAGAAGCAGGTTGAGATCGCCAGCAAAGTGGACCGTGTTTACGATTTTGCCCTGCCGCCGCTGCTGCTGCATAGTCTGTTTACCGGTCATGTTGAACCGGTTGCACATTGGACCGAAATTCGTCCGAATAATGCAGTTACCGTTCTGGATACCCATGATGGTATTGGTGTTATTGATATCGGCAGCGACCAGCTGGATCGTAGCCTGAAAGGTCTGGTTCCGGATGAAGATGTTGATAATCTGGTTA ATACCATCCACGCCAACACCCATGGTGAGAGCCAGGCAGCAACAGGTGCAGCAGCAAGTAATCTGGATCTGTATCAGGTTAATAGCACCTATTATAGCGCCCTGGGTTGTAATGATCAGCATTATCTGGCAGCACGTGCAGTTCAGTTTTTTCTGCCGGGTGTTCCGCAGGTTTATTATGTTGGTGCACTGGCAGGTCGTAATGATATGGAACTGCTGCGTCGTACCAATAATGGTCGTGATATT AAT CGCCATTACTACAGCACCGCAGAAATTGATGAAAACCTGGAACGTCCGGTTGTTAAAGCACTGAATGCACTGGCAAAATTTCGCAATGAACTGCCGGCATTTGATGGTGAATTTAGCTATGAAGTTGACGGCGACACCAGCATTACCTTTCGTTGGACCGCAGCAGATGGTACCAGCACCGCAGCATTAACATTTGAACCGGGTCGTGGTCTGGGTACCGATAATGCAACCCCGGTTGCAAGCCTGGCATGGAGCGATGCAGCAGGTGATCATGAAACCCGTGATCTGCTGGCAAATCCGCCGATTGCAGATATTGAT (where the bold underlined part indicates the mutation site).

[0027] In this invention, the gene encoding the mutant sucrose phosphorylase was obtained by mutation based on AAO84039.1, and the specific codons of the mutation sites are shown in Table 1.

[0028] This invention provides the application of the mutant sucrose phosphorylase in the catalytic synthesis of L-ascorbic acid 2-O-α-D-glucoside.

[0029] In this invention, a reaction system is obtained by mixing sucrose, mutant sucrose phosphorylase and L-ascorbic acid, and L-ascorbic acid 2-O-α-D-glucoside is prepared by enzyme-catalyzed reaction.

[0030] In this invention, the concentration of L-ascorbic acid in the reaction system is 0.2~2.6 mol / L, preferably 0.5~2.2 mol / L, more preferably 1.2~2.0 mol / L. The concentration of sucrose in the reaction system is preferably 1~8 mol / L, more preferably 1.2~5 mol / L, and most preferably 1.3~1.8 mol / L.

[0031] In this invention, the mutant sucrose phosphorylase is preferably used in the form of a whole-cell catalyst, and the concentration of the mutant sucrose phosphorylase in the bacterial solution is preferably 0.5~6 mg / mL, more preferably 1~4 mg / mL, and most preferably 1~2 mg / mL.

[0032] In this invention, the preparation method of the mutant sucrose phosphorylase whole-cell catalyst includes the following steps: 1) transferring the pET28a plasmid carrying the DNA molecule encoding the mutant sucrose phosphorylase into *E. coli* for expression; 2) collecting the precipitate by centrifugation to obtain the whole-cell catalyst. This invention does not specifically limit the method and parameters for transferring the DNA molecule into *E. coli* for expression; conventional methods for exogenous gene transfer and expression in the art can be used. In the specific implementation of this invention, it is preferred to use PCR to mutate and amplify the pET28a plasmid containing the wild-type sucrose phosphorylase gene, obtain the pET28a plasmid containing the mutant sucrose phosphorylase DNA molecule, and transfer it into *E. coli* for protein expression; then, it is preferred to collect the bacterial cells by freeze centrifugation, and the precipitate is the whole-cell catalyst.

[0033] In this invention, the temperature of the enzyme-catalyzed reaction is preferably 20℃~60℃, more preferably 30℃~50℃; the time of the enzyme-catalyzed reaction is preferably 12 h~72 h, more preferably 24 h~60 h; the pH value of the enzyme-catalyzed reaction is preferably 4~6; the volume of the reaction system is preferably 5~2000L, more preferably 10~1000L, and most preferably 100~800L.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] This embodiment constructs a sucrose phosphorylase mutant. The NNK saturation mutagenesis strategy was employed, using the pET28a plasmid containing the wild-type sucrose phosphorylase gene as a DNA template. Point mutation primers were designed using SnapGene software. The PCR system consisted of: 1.2 μL upstream primer, 1.2 μL downstream primer, 1 μL plasmid, 11.6 μL ultrapure water, and 15 μL Primer Star Max. The PCR program was: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 70 s, for 29 cycles. The PCR product was digested with Dpn I, and the digested product was recovered by gel electrophoresis and transfected into *E. coli* BL21(DE3) competent cells. After kanamycin resistance screening and mutation site sequencing verification, recombinant *E. coli* BL21(DE3) expressing the mutant sucrose phosphorylase was obtained.

[0037] The mutation sites of mutant sucrose phosphorylase and their corresponding amino acid and nucleotide sequences are shown in Table 1.

[0038] Table 1. Amino acid and nucleotide sequences of the mutation sites.

[0039] Example 2

[0040] In this embodiment, the optimal temperatures for wild-type sucrose phosphorylase (WT) and different mutant sucrose phosphorylases were determined. The specific method was as follows: 500 μL of 1 mg / mL mutant sucrose phosphorylase, 200 μL of deionized water, 150 μL of 1M L-ascorbic acid, and 150 μL of 1.2M sucrose were added to the reaction system, and the reaction was carried out in metal baths at 20℃, 30℃, 40℃, 50℃, and 60℃ for 2 h, respectively. The reaction was terminated at 100℃ for 10 min, and the optimal temperatures for wild-type and mutant sucrose phosphorylases were determined.

[0041] The mutant sucrose phosphorylases are as follows: A84G, G147L, V233L, Y237F, K238D, N402A, A84G / V233L, A84G / G147L, A84G / N402A, V233L / Y237F, V233L / K238D, V233L / N402A, Y237F / N402A, K238D / N402A. A84G / V233L / Y237F, G147L / K238D / N402A, V233L / K238D / N402A, Y237F / K238D / N402A, G147L / Y237F / K238D, A84G / G147L / V233L / Y237F, A84G / G147L / V233L / K238D, G147L / V233L / K238D / N402A, V233L / Y2 37F / K238D / N402A, A84G / G147L / V233L / Y237F / K238D, A84G / G147L / V233L / Y237F / N402A, A84G / V233 L / Y237F / K238D / N402A, G147L / V233L / Y237F / K238D / N402A, A84G / G147L / V233L / Y237F / K238D / N402A The results are as follows Figures 1-2 As shown in Table 2, the peak area of ​​L-ascorbic acid 2-O-α-D-glucoside in the supernatant was detected by HPLC (10 mM phosphate, UV detector, C18 column, flow rate 0.5 mL / min, column temperature 40 ℃). The relative activities of wild-type and different mutant sucrose phosphorylases at different temperatures were calculated based on the peak area of ​​the products. The optimal temperature of WT was 50 ℃, and the optimal temperature of A84G / G147L / V233L / Y237F was 40 ℃. The optimal temperature of sucrose phosphorylase changed significantly after mutation.

[0042] Table 2. Optimal temperatures for wild-type and mutant sucrose phosphorylases

[0043] Example 3

[0044] This embodiment measures the yield of L-ascorbic acid 2-O-α-D-glucoside prepared by wild-type sucrose phosphorylase (WT) and mutant sucrose phosphorylase at high substrate concentrations. The specific method is as follows: 1.5 L of OD was added to a 5 L enzymatic hydrolysis reactor. 600A whole-cell catalyst for a mutant sucrose phosphorylase with a concentration of 60 was used, with 1.4 M sucrose, 1.8 M L-ascorbic acid, and a total reaction volume of 3 L. The reaction was carried out at the optimum temperature (determined in Table 2 of Example 2) for 48 h, and terminated at 100 °C for 10 min. The peak area of ​​L-ascorbic acid 2-O-α-D-glucoside in the supernatant was detected by HPLC (10 mM phosphate, UV detector, C18 column, flow rate 0.5 mL / min, column temperature 40 °C). The yields of wild-type and different mutant sucrose phosphorylases under their optimum reaction conditions were calculated based on the peak area of ​​the products. As shown in Table 3, the highest yield of L-ascorbic acid 2-O-α-D-glucoside was obtained by the catalyst A84G / G147L / V233L / Y237F, which was 427 g / L, 3.19 times that of WT.

[0045] The mutant sucrose phosphorylases are as follows: A84G, G147L, V233L, Y237F, K238D, N402A, A84G / V233L, A84G / G147L, A84G / N402A, V233L / Y237F, V233L / K238D, V233L / N402A, Y237F / N402A, K238D / N402A, A84G / V233L / Y237F, G147L / K238D / N402A, V233L / K238D / N402A, Y237F / K238D / N402A, G147L / Y237F / K238D, A84G / G14 7L / V233L / Y237F, A84G / G147L / V233L / K238D, G147L / V233L / K238D / N40 2A, V233L / Y237F / K238D / N402A, A84G / G147L / V233L / Y237F / K238D, A84 G / G147L / V233L / Y237F / N402A, A84G / V233L / Y237F / K238D / N402A, G147 L / V233L / Y237F / K238D / N402A, A84G / G147L / V233L / Y237F / K238D / N402A

[0046] Table 3 Comparison of catalytic yields of wild-type and mutant sucrose phosphorylases

[0047] Example 4

[0048] This embodiment measures the yield of L-ascorbic acid 2-O-α-D-glucoside prepared by wild-type sucrose phosphorylase (WT), mutant sucrose phosphorylases A84G, Y237F, A84G / V233L, A84G / N402A, V233L / Y237F, A84G / G147L / V233L / Y237F, A84G / G147L / V233L / Y237F / K238D, and A84G / G147L / V233L / Y237F / K238D / N402A in a larger reaction system. The specific method is the same as in Example 3, except that the bioreactor capacity is increased from 5 L to 1000 L and the reaction system is increased from 3 L to 600 L.

[0049] The results are shown in Table 4. The highest yield of L-ascorbic acid 2-O-α-D-glucoside was obtained by catalysis with A84G / G147L / V233L / Y237F, which was 435 g / L, 3.13 times that of WT.

[0050] Table 4. Comparison of large-scale catalytic yields of wild-type and mutant sucrose phosphorylases

[0051] As can be seen from the above embodiments, the various mutant sucrose phosphorylases provided by the present invention exhibit significant improvements in catalytic activity compared to wild-type sucrose phosphorylases. In particular, the mutant sucrose phosphorylases A84G / G147L / V233L / Y237F possess high catalytic activity and can achieve high-yield preparation of L-ascorbic acid 2-O-α-D-glucoside at extremely high substrate concentrations, showing broad application prospects.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant sucrose phosphorylase, characterized in that, The mutant sucrose phosphorylase is obtained by point mutation of wild-type sucrose phosphorylase, and the amino acid sequence of the wild-type sucrose phosphorylase is shown in SEQ ID NO.1; the mutant sucrose phosphorylase contains one or more of the following site mutations: A84G, G147L, V233L, Y237F, K238D, N402A.

2. The mutant sucrose phosphorylase according to claim 1, characterized in that, The mutant sucrose phosphorylase contains one of the following two-site combination mutations: A84G / V233L, A84G / G147L, A84G / N402A, V233L / Y237F, V233L / K238D, V233L / N402A, Y237F / N402A, or K238D / N402A.

3. The mutant sucrose phosphorylase according to claim 1, characterized in that, The mutant sucrose phosphorylase contains one of the following three-point combination mutations: A84G / V233L / Y237F, G147L / K238D / N402A, V233L / K238D / N402A, Y237F / K238D / N402A, or G147L / Y237F / K238D.

4. The mutant sucrose phosphorylase according to claim 1, characterized in that, The mutant sucrose phosphorylase contains one of the following four-site combination mutations: A84G / G147L / V233L / Y237F, A84G / G147L / V233L / K238D, G147L / V233L / K238D / N402A, V233L / Y237F / K238D / N402A.

5. The mutant sucrose phosphorylase according to claim 1, characterized in that, The mutant sucrose phosphorylase contains one of the following five-site combination mutations: A84G / G147L / V233L / Y237F / K238D, A84G / G147L / V233L / Y237F / N402A, A84G / V233L / Y237F / K238D / N402A, G147L / V233L / Y237F / K238D / N402A.

6. The mutant sucrose phosphorylase according to claim 1, characterized in that, The mutant sucrose phosphorylase contains the following six-site combination mutations: A84G / G147L / V233L / Y237F / K238D / N402A.

7. A DNA molecule encoding the mutant sucrose phosphorylase according to any one of claims 1 to 6.

8. The use of the mutant sucrose phosphorylase according to any one of claims 1 to 6 in the catalytic synthesis of L-ascorbic acid 2-O-α-D-glucoside.

9. The application according to claim 8, characterized in that, A reaction system was obtained by mixing sucrose, mutant sucrose phosphorylase and L-ascorbic acid, and L-ascorbic acid 2-O-α-D-glucoside was prepared by enzyme-catalyzed reaction. The concentration of L-ascorbic acid in the reaction system is 0.2~2.6 mol / L, the mutant sucrose phosphorylase is used in the form of a whole-cell catalyst, and the concentration of mutant sucrose phosphorylase in the whole-cell catalyst is 0.5~6 mg / mL; the concentration of sucrose in the reaction system is 1~8 mol / L.

10. The application according to claim 9, characterized in that, The enzyme-catalyzed reaction is carried out at a temperature of 20℃ to 60℃, for a time of 12 h to 72 h, and at a pH of 4 to 6; the reaction system has a volume of 5 to 2000 L.

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