Mutant enzymes of sucrose phosphorylase, glucose isomerase and kojibiose synthase, fermentation production method thereof and application in synthesis of kojibiose

Abstract

The present application discloses mutant enzymes of sucrose phosphorylase, glucose isomerase and kojibiose synthase, fermentation production methods thereof and applications in synthesizing kojibiose. The amino acid sequence of the sucrose phosphorylase mutant enzyme is shown in SEQ.ID.NO.1, the amino acid sequence of the glucose isomerase mutant enzyme is shown in SEQ.ID.NO.3, and the amino acid sequence of the kojibiose synthase mutant enzyme is shown in SEQ.ID.NO.5. The method for synthesizing kojibiose comprises the following steps: adding sucrose phosphorylase mutant enzyme, glucose isomerase mutant enzyme and kojibiose synthase mutant enzyme to sucrose, heating the solution to 40°C and stirring slightly, and completing the reaction after at least 4 hours. Based on the two phosphorylases, the present invention further improves the substrate selectivity and product generation direction through the transformation of specific properties. At the same time, glucose isomerase is used to carry out the mutual conversion between fructose and glucose, so as to realize the effective conversion between sucrose and kojibiose.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and specifically relates to mutant enzymes of sucrose phosphorylase, glucose isomerase and kojibiose synthase, a fermentation production method thereof, and an application of the mutant enzymes in synthesizing kojibiose. Background Art

[0002] α-Gluco-α-glucoside, also known as Kojibiose, is a disaccharide product of glucose caramelization; it exists in honey in nature. Kojibiose is an ideal substitute for traditional edible sugars such as sucrose and fructose. It has a mild sweetness, contains fewer calories, can increase iron absorption, and can prevent tooth decay. It is also a prebiotic. At the same time, Kojibiose is also an inhibitor of plant glucosidase I and can be used to help identify and characterize glucosidase I enzymes involved in the terminal deglycosylation of high-mannose oligosaccharides. Kojibiose is a growth factor for bifidobacteria, lactic acid bacteria, and true bacteria. Kojibiose has antitoxic activity and can reduce the liver expression of inflammatory markers in the body.

[0003] Due to its low abundance in nature, kojibiose cannot be produced on a large scale by separation; at the same time, industrial chemical synthesis of kojibiose is also very difficult. Therefore, the development of a scalable and inexpensive biological preparation method is of great significance for the market promotion and application of this disaccharide. Summary of the invention

[0004] The present invention provides mutant enzymes of sucrose phosphorylase, glucose isomerase and kojibiose synthase, a fermentation production method thereof and an application of the mutant enzymes in synthesizing kojibiose.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The sucrose phosphorylase mutant enzyme (SucPhy), whose amino acid sequence is shown in SEQ.ID.NO.1, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.2.

[0007] The sucrose phosphorylase mutant enzyme is a natural (or wild type) sucrose phosphorylase (WT SucPhy) (Uniprot ID: W5IHN4) derived from the rare Scardovia inopinata, with the following site mutations: D121M, V194W, L341I, Q345S, S352T, E410Q. The modified mutant enzyme has a high kojibiose synthase function while retaining its original sucrose phosphorylase function.

[0008] Glucose isomerase mutant enzyme (BtIso), whose amino acid sequence is shown in SEQ.ID.NO.3, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.4.

[0009] The glucose isomerase mutant enzyme is a natural glucose isomerase mutant enzyme (WTBtIso) (Uniprot ID: Q8A9M2, EC5.3.1.5) derived from Bacteroides thetaiotaomicron, with the following site mutations: N72S, K122N, H149C. The activity and catalytic stability are further improved through mutation modification.

[0010] The kojibiose synthase mutant enzyme (KojiPhy), whose amino acid sequence is shown in SEQ.ID.NO.5, and whose encoding nucleotide sequence is shown in SEQ.ID.NO.6.

[0011] The kojibiose synthase mutant enzyme is a natural kojibiose synthase (WTKojiPhy) (Uniprot ID: Q8L163, EC 2.4.1.230) derived from thermoanaerobacter brockii, with the following site mutations: V183N, K184E, F288G, P369H, I372S, V374T, W555D. The modification not only improves its activity and stability, but also significantly improves product generation.

[0012] The fermentation production method of the mutant enzyme comprises the following steps:

[0013] (1) synthesizing the nucleotide sequence shown in SEQ.ID.NO.2, SEQ.ID.NO.4 or SEQ.ID.NO.6, and then subcloning it into the pET28a plasmid through the NdeI / XhoI restriction site, and then transferring the plasmid into Escherichia coli cells for plate culture, and finally selecting a single clone for liquid step-by-step amplification culture;

[0014] (2) Transfer the single colony on the plate into LB culture medium containing more than 50 μM kanamycin for culture. When the cells grow to the logarithmic phase, inoculate them into LB culture medium containing the same antibiotic, and finally transfer them to a fermenter for culture. When the OD value of the fermentation liquid reaches 20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to induce protein expression for at least 6 hours, and then collect the wet cells by centrifugation.

[0015] (3) The wet cells were mixed with the buffer solution, and then the cells were broken by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell wall, and finally the enzyme-containing clear solution was obtained;

[0016] The pH value of the buffer in step (3) is 7.0-9.0, preferably 50 mM Tris-HCl buffer.

[0017] The use of the above-mentioned sucrose phosphorylase mutant enzyme (SucPhy), glucose isomerase mutant enzyme (BtIso) or kojibiose synthase mutant enzyme (KojiPhy) in the synthesis of kojibiose.

[0018] A method for synthesizing kojibiose comprises the following steps:

[0019] Sucrose is added to the buffer, followed by sucrose phosphorylase mutant enzyme (SucPhy), glucose isomerase mutant enzyme (BtIso) and kojibiose synthase mutant enzyme (KojiPhy), the solution is heated to 35-50°C and gently stirred, and the pH value of the reaction system is maintained at 6.5-7.5 during the reaction; the reaction is completed after at least 4 hours, and the enzyme is inactivated.

[0020] The synthetic raw material may also include glucose.

[0021] In the method, the enzyme can be immobilized and the multiple enzyme system can be recycled multiple times, thereby further reducing the production price and improving the convenience of the production process;

[0022] After the enzyme is inactivated, kojibiose solid is obtained by separation and purification.

[0023] The pH value of the buffer solution is 7.0-9.0; the buffer solution is preferably a phosphate buffer solution with a pH of 7.0.

[0024] The enzyme immobilization comprises the following steps:

[0025] Sucrose phosphorylase mutant enzyme (SucPhy), glucose isomerase mutant enzyme (BtIso) and kojibiose synthase mutant enzyme (KojiPhy) were dissolved in a buffer solution at an activity unit ratio of 1:(1.8-2.5):(1.5-2.2), followed by adding phenoxyacetic acid and epoxy resin, stirring at room temperature for at least 6 hours, filtering out the immobilized enzyme, and finally washing with water and buffer solution three times each, and drying for use;

[0026] In the method, the ratio of enzyme to epoxy resin is (5-20) U:1 g;

[0027] The pH value of the buffer solution is 8.0, preferably 50 mM potassium phosphate solution or 25 mM phosphate buffer;

[0028] The epoxy resin is preferably LX-1000EP epoxy resin.

[0029] The separation and purification comprises the following steps:

[0030] Yeast is added to the reaction solution, and aerobic fermentation is carried out at 25-32°C for at least 10 hours to remove residual disaccharides and monosaccharides. After the fermentation is completed, the supernatant is collected by centrifugation, and then the pH value is adjusted back to 6.5-8.0, and then anion exchange resin is used to remove phosphoric acid impurities, and finally, a reverse osmosis membrane is used to remove salts, concentrate, and crystallize to obtain kojibiose solid.

[0031] Compared with the prior art, the present invention has the following advantages and effects:

[0032] 1. Kojibiose is the combination of two glucose molecules at the 1,2-glycosidic bond. Sucrose is the combination of one glucose molecule and one fructose molecule at the 1,4-glycosidic bond. Sucrose phosphorylase (EC: 2.4.1.7) can use monophosphate to catalyze the conversion of sucrose, glucose-1-phosphate and fructose, while kojibiose phosphorylase (EC: 2.4.1.230) can use monophosphate to catalyze the conversion of kojibiose, glucose-1-phosphate and glucose. Based on the above two phosphorylases, the present invention can further improve its substrate selectivity and product generation direction through the modification of specific properties. At the same time, in combination with glucose isomerase, it can convert fructose and glucose, so that the effective conversion between sucrose and kojibiose can be achieved.

[0033] 2. The present invention provides a fully enzymatic preparation process for kojibiose, the overall process of which is to use sucrose as the starting material, generate glucose-1-phosphate and fructose (Fructose) in sucrose phosphorylase (SucPhy), then convert fructose into glucose under the action of isomerase BtIso, and finally connect glucose-1-phosphate and glucose into kojibiose under the action of kojibiose synthase (KojiPhy). This route is very simple, has a high conversion rate, and the raw materials used are also very cheap. The conversion can be achieved once with a liquid enzyme, or it can be achieved by using an immobilized enzyme. Therefore, the patented synthetic kojibiose preparation process has many advantages such as being green, cheap, and easy to scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The SDS-PAGE gel detection images of the prepared enzymes: 1) KojiPhy, 2) SucPhy, 3) BtIso.

[0035] Figure 2 Kojibiose 1 H-NMR spectrum. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0037] Example 1: Enzyme modification

[0038] Sucrose phosphorylase mutant enzyme (SucPhy): Wild-type sucrose phosphorylase (WTSucPhy) (Uniprot ID: W5IHN4) is derived from the rare Scardovia inopinata. Its original activity is sucrose phosphorylase, and it also has weak kojibiose synthase function. After comprehensive mutation transformation, it has high kojibiose synthase function while retaining its original sucrose phosphorylase function. Its mutation sites are: D121M, V194W, L341I, Q345S, S352T, E410Q.

[0039] Glucose isomerase mutant enzyme (BtIso): Wild-type glucose isomerase (WTBtIso) (Uniprot ID: Q8A9M2, EC 5.3.1.5) is derived from Bacteroides thetaiotaomicron. It has natural isomerization activity to xylose and glucose. After mutation modification, its activity and catalytic stability are further improved. Its mutation sites are: N72S, K122N, H149C;

[0040] Kojibiose synthase mutant enzyme (KojiPhy): Wild-type Kojibiose synthase (WTKojiPhy) (Uniprot ID: Q8L163, EC 2.4.1.230) is derived from Thermoanaerobacter brockii. The enzyme has a relatively high activity in the decomposition and synthesis of Kojibiose. On this basis, the enzyme was modified to improve not only its activity and stability, but also the product generation. The final mutation sites are: V183N, K184E, F288G, P369H, I372S, V374T, W555D.

[0041] The basic idea of ​​transformation is to achieve enzyme amino acid site replacement by rationally / randomly replacing the gene template bases corresponding to the modified enzyme, thereby ultimately changing the performance of the enzyme. In the practice of this patent, firstly, a homologous multiple sequence alignment (Multiple Sequence Alignment) will be performed on the SucPhy, BtIso, and Kojiphy enzymes to preliminarily determine the overall analysis of their family amino acid conserved sites, and then the Discovery Studio commercial software will be used to perform homologous modeling and ligand docking on each enzyme to ultimately determine the substrate binding site; under the guidance of the above two aspects, site-directed mutagenesis tests are performed to improve the catalytic activity of its substrate; at the same time, error-prone PCR high-throughput screening experiments can be used to locate remote sites to be replaced, thereby guiding the experiment to improve the activity and stability of the enzyme. Finally, through the integration of the above aspects, enzyme mutants with improved performance in all aspects were successfully obtained.

[0042] Example 2: Fermentation production of enzymes

[0043] (1) Synthesize the nucleotide sequence shown in SEQ.ID.NO.2, SEQ.ID.NO.4 or SEQ.ID.NO.6, and then subclone it into the pET28a plasmid through the NdeI / XhoI restriction site, and transfer the plasmid into E.coli (BL21) (Qingke Biotechnology) cells for plate culture, and finally select a single clone for liquid step-by-step amplification culture.

[0044] (2) First, transfer the single colony on the plate into 5 mL of LB culture medium containing 50 μM kanamycin (37°C) for culture. When the cells grow to the logarithmic phase, inoculate them into 250 mL of LB culture medium containing the same antibiotic, and finally transfer them to a 5 L culture fermenter for culture. When the OD value of the fermentation liquid is 20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 25°C to induce protein expression for 6 hours, and then centrifuge (4000 rpm, 15 min) to collect 20-30 g of wet cells.

[0045] (3) To verify the expression of the enzyme, a small amount of cells was first mixed with Tris-HCl buffer (50 mM, pH 8.0), and then the cells were broken by freeze-thaw method. After high-speed centrifugation, the supernatant was run on SDS-PAGE protein gel (sodium dodecyl sulfate-polyacrylamide gel) to determine the soluble expression of the protein; the results are as follows Figure 1 shown.

[0046] The remaining cells confirmed to be correct are first mixed with the buffer (10 grams of wet cells are mixed with about 200 mL of buffer), and then the cells are crushed by high pressure and centrifuged at high speed (16000 rpm, 45 min) to remove the cell wall. The enzyme-containing clear liquid obtained can be used directly in subsequent use (the liquid enzyme activity is 200-350 U / mL, U is the amount of enzyme required to convert 1 μmol of substrate in one minute at room temperature) or further purified and immobilized for use (solid enzyme reaction).

[0047] The LB medium consists of 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% dipotassium hydrogen phosphate and 5% glycerol.

[0048] Mixed immobilization of enzymes:

[0049] Ammonium sulfate solid was gradually added to the crude enzyme solutions of sucrose phosphorylase (SucPhy), glucose isomerase (BtIso) from Bacteroides thetaiotaomicron, and kojibiose synthase (KojiPhy) collected above until the enzyme precipitated (40%-60%, w / v ammonium sulfate / buffer). The enzyme solid was then collected by centrifugation (10000rpm, 12min) and slowly dissolved in 25mM Tris buffer at pH 8.0. Finally, the enzyme was desalted by G25 size exclusion chromatography column (purchased from Sigma) and separated by DEAE Seplite FF (Xi'an Lanxiao Company) anion exchange column to obtain the initially purified liquid enzymes SucPhy, BtIso, and KojiPhy. The enzyme solution can be directly used for subsequent enzyme fixation. In the mixed fixation of SucPhy / BtIso / KojiPhy enzymes, the initially purified enzymes were mixed and fixed using LX-1000EP epoxy resin (Xi'an Lanxiao Company) according to the activity unit ratio of 1:2:2. The basic method of immobilization is: 5000U of mixed enzyme mixed according to the above activity unit ratio is dissolved in 2L 50mM pH 8.0 potassium phosphate solution, then 40mM phenoxyacetic acid and 600g LX-1000EP epoxy resin are added to the buffer solution, and the immobilized enzyme is filtered out after stirring at room temperature for 6 hours, and finally washed three times with clean water and 25mM pH 8.0 phosphate buffer, and then dried at low temperature for use; the SucPhy / BtIso / KojiPhy immobilized mixed enzyme has 75-81% of the activity of the corresponding liquid enzyme.

[0050] Example 3: Using sucrose as raw material, liquid enzymes (SucPhy, BtIso, KojiPhy) to prepare kojibiose in one go

[0051]

[0052] 68.4 g of sucrose (200 mM) was added to 1 L of 25 mM pH 7.0 phosphate buffer, and then 1000 U of SucPhy crude enzyme solution, 1500 U of BtIso crude enzyme solution and 2000 U of KojiPhy crude enzyme solution were added at once. The solution was heated to 40 ° C and stirred slightly. The reaction pH was always maintained at about 6.5-7.5 during the reaction. After 4 hours, the reaction was completed, and then the solution was heated to 90 ° C for 20 minutes to inactivate the enzyme. Finally, 30 g of baker's yeast was added to the reaction solution for aerobic fermentation at 30 ° C for 10 hours to remove residual sucrose and glucose. After the fermentation was completed, the supernatant was collected by centrifugation, and then the pH value was adjusted back to 7.0 and the phosphoric acid impurities were removed by D201 anion exchange resin. Finally, the reverse osmosis membrane was used for desalination, concentration, and crystallization (ethanol: H2O = 2: 1, V: V) to obtain 37 g of white granular solid (54% yield).

[0053] Example 4: Using sucrose and glucose as raw materials, liquid enzymes (SucPhy, BtIso, KojiPhy) to prepare kojibiose at one time

[0054]

[0055] Similar to Example 1, but glucose was added into the reaction solution at the same time.

[0056] 68.4 g of sucrose (200 mM) and 18 g of glucose (100 mM) were added to 1 L of 25 mM pH 7.0 phosphate buffer, and then 1000 U of SucPhy crude enzyme solution, 1500 U of BtIso crude enzyme solution and 2000 U of KojiPhy crude enzyme solution were added at once. The solution was heated to 40 ° C and stirred slightly. The reaction pH was always maintained at about 6.5-7.5 during the reaction. After 4 hours, the reaction was completed, and then the solution was heated to 90 ° C and maintained for 20 minutes to inactivate the enzyme. Finally, 30 g of baker's yeast was added to the reaction solution for aerobic fermentation at 30 ° C for 10 hours to remove residual sucrose and glucose. After fermentation, the supernatant was collected by centrifugation, and then the pH value was adjusted back to 7.0 and the phosphoric acid impurities were removed by D201 anion exchange resin. Finally, the reverse osmosis membrane was used for desalination, concentration, and crystallization (ethanol: H2O = 2: 1, V: V) to obtain 57 g of white granular solid (83% yield).

[0057] Example 5: Preparation of kojibiose by immobilized mixed enzyme using sucrose and glucose as raw materials

[0058]

[0059] The reaction is similar to the above embodiment, but the immobilized enzyme can be recovered and reused later.

[0060] 68.4 g of sucrose (200 mM) and 18 g of glucose (100 mM) were added to 1 L of 25 mM pH 7.0 phosphate buffer, and then 5000 U of immobilized mixed enzyme was added at one time. The solution was heated to 50 ° C and stirred slightly. The reaction pH was always maintained at about 6.5-7.5 during the reaction. After 8 hours, the reaction was completed, and then the immobilized mixed enzyme was collected by filtration (the mixed enzyme retained 80% of its initial activity after 10 uses). Similar to the above treatment, 30 g of baker's yeast was added to the reaction solution for aerobic fermentation at 30 ° C for 10 hours to remove residual sucrose and glucose. After the fermentation was completed, the supernatant was collected by centrifugation, and then its pH value was adjusted back to 7.0 and phosphoric acid impurities were removed using D201 anion exchange resin. Finally, it was desalted using a reverse osmosis membrane, concentrated, and crystallized (ethanol: H2O = 2:1, V:V) to obtain 60 g of white granular solid (88% yield).

[0061] Comparative Example: Using sucrose and glucose as raw materials, natural liquid enzymes (WT SucPhy, WTBtIso, WTKojiPhy) were used to prepare kojibiose in one go

[0062]

[0063] The reaction conditions are similar to those in Example 2, but the enzyme used is the natural enzyme (WT)

[0064] 68.4 g of sucrose (200 mM) and 18 g of glucose (100 mM) were added to 1 L of 25 mM pH 7.0 phosphate buffer, and then 3000 U of WT SucPhy crude enzyme solution, 2500 U of WT T Iso crude enzyme solution and 5000 U of WT KojiPhy crude enzyme solution were added at once. The solution was heated to 40 ° C and stirred slightly. The reaction pH was always maintained at about 6.5-7.5 during the reaction. After 8 hours, the reaction was completed, and then the solution was heated to 90 ° C for 20 minutes to inactivate the enzyme. Finally, 50 g of baker's yeast was added to the reaction solution for aerobic fermentation at 30 ° C for 24 hours to remove residual sucrose and glucose. After fermentation, the supernatant was collected by centrifugation, and then the pH value was adjusted back to 7.0 and the phosphoric acid impurities were removed by D201 anion exchange resin. Finally, the reverse osmosis membrane was used for desalination, concentration, and crystallization (ethanol: H2O = 2: 1, V: V) to obtain 14 g of off-white solid (21% yield).

[0065] The solid kojibiose obtained in each example and comparative example was subjected to hydrogen nuclear magnetic resonance spectroscopy, with D2O as solvent and Varian 600 MHz nuclear magnetic resonance. The results are as follows: Figure 2 As shown, it is proved that the product is kojibiose.

[0066] Table 1: Performance comparison of the mutant enzyme of the present invention and its wild-type enzyme

[0067]

[0068] Note: The expression levels of the enzymes were determined after fermentation according to the method of Example 2.

[0069] It can be seen from the results of Table 1, the embodiments and the comparative examples that, compared with the corresponding wild enzyme, the activity, thermal stability and expression level of the mutant enzyme of the present invention are significantly improved; and the yield is greatly increased when used for synthesizing kojibiose.

[0070] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A sucrose phosphorylase mutant enzyme, characterized in that: The amino acid sequence is shown in SEQ ID NO.1, and the encoding nucleotide sequence is shown in SEQ ID NO.

2.

2. A sucrose phosphorylase mutant enzyme, characterized in that: The following site mutations were made to the natural sucrose phosphorylase: D121M, V194W, L341I, Q345S, S352T, E410Q; The Uniprot ID of the natural sucrose phosphorylase is W5IHN4.

3. A glucose isomerase mutant enzyme, characterized in that: The amino acid sequence is shown in SEQ ID NO.3, and the encoding nucleotide sequence is shown in SEQ ID NO.

4.

4. A glucose isomerase mutant enzyme, characterized in that: The following site mutations were made to the natural glucose isomerase: N72S, K122N, H149C; The Uniprot ID of the natural glucose isomerase is Q8A9M2.

5. A mutant enzyme of kojibiose synthase, characterized in that: The amino acid sequence is shown in SEQ ID NO.5, and the encoding nucleotide sequence is shown in SEQ ID NO.

6.

6. A mutant enzyme of kojibiose synthase, characterized in that: The following site mutations were made to the native kojibiose synthase: V183N, K184E, F288G, P369H, I372S, V374T, W555D; The Uniprot ID of the natural kojibiose synthase is Q8L163.

7. A fermentation production method of the sucrose phosphorylase mutant enzyme according to claim 1 or 2, the glucose isomerase mutant enzyme according to claim 3 or 4, or the kojibiose synthase mutant enzyme according to claim 5 or 6, characterized in that The following steps are involved: (1) synthesizing the nucleotide sequence shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.6, and then subcloning it into the pET28a plasmid through the NdeI / XhoI restriction site, and then transferring the plasmid into Escherichia coli cells for plate culture, and finally selecting a single clone for liquid step-by-step amplification culture; (2) Transfer the single colony on the plate into LB culture medium containing 50 μM kanamycin for culture. When the cells grow to the logarithmic phase, inoculate them into LB culture medium containing the same antibiotic, and finally transfer them to a fermenter for culture. When the OD value of the fermentation liquid reaches 20, add 0.5 mM isopropyl-β-D-thiogalactopyranoside to induce protein expression for at least 6 hours, and then collect the wet cells by centrifugation. (3) The wet cells are mixed with the buffer solution, and then the cells are broken by high pressure and centrifuged at high speed to remove the cell walls, and finally the enzyme-containing clear solution is obtained.

8. Use of the sucrose phosphorylase mutant enzyme according to claim 1 or 2, the glucose isomerase mutant enzyme according to claim 3 or 4, or the kojibiose synthase mutant enzyme according to claim 5 or 6 in synthesizing kojibiose.

9. A method for synthesizing kojibiose, characterized in that The following steps are involved: Sucrose is added to the buffer, and then the sucrose phosphorylase mutant enzyme according to claim 1 or 2, the glucose isomerase mutant enzyme according to claim 3 or 4, and the kojibiose synthase mutant enzyme according to claim 5 or 6 are added, the solution is heated to 35-50°C and gently stirred, and the pH value of the reaction system is maintained at 6.5-7.5 during the reaction; the reaction is completed after at least 4 hours, and the enzyme is inactivated.

10. The method according to claim 9, characterized in that: The synthetic raw material also includes glucose.

11. The method according to claim 9, characterized in that: The enzyme is immobilized for use.

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