Micellar multienzyme compound based on block polymer as well as preparation method and application of micelle multienzyme compound

Abstract

The invention discloses a micelle multienzyme compound based on a block polymer as well as a preparation method and application thereof.The cyclodextrin-b-polyethylene glycol-b-polystyrene block polymer is adopted to wrap multienzyme molecules, nano-micelles are constructed, multienzyme is embedded through the polymer to form a nano confinement catalytic system, and the micelle multienzyme compound based on the block polymer is prepared. The multi-enzyme cascade catalytic efficiency and stability are obviously improved. When the micelle multienzyme compound is used as a catalyst to catalyze maltodextrin to synthesize D-psicose, the catalytic performance of the micelle multienzyme compound is obviously superior to that of a free multienzyme catalytic system, and the micelle multienzyme compound has important significance in promoting industrial application of a multienzyme biological catalysis technology.

Description

(1) Technical field

[0001] The present invention belongs to the field of enzyme engineering, and in particular relates to a micellar multi-enzyme complex based on block polymers, a preparation method and an application thereof. (2) Background technology

[0002] Block polymers are polymer chains formed by the polymerization of two or more different types of monomers. Their structure consists of different polymer segments (such as hydrophilic and hydrophobic segments) connected by covalent bonds. Depending on the arrangement of the segments, block polymers can be classified into various morphologies, including linear, star-shaped, and branched. This structural diversity gives block polymers broad potential for application in fields such as biochemistry.

[0003] Block polymers self-assemble in solution to form specific micellar structures. These structures possess well-defined nanoscale spaces that restrict the movement of enzyme molecules by encapsulation, thereby producing a confinement effect. This approach prevents enzyme leakage through physical confinement, preserving the enzyme's three-dimensional structure and catalytic activity to the greatest extent possible. The unique molecular structure of block polymers (such as the alternating arrangement of hydrophobic and hydrophilic blocks) can form a stable microenvironment and construct a nanoscale confined space, thereby effectively reducing the diffusion of enzyme molecules, enhancing the interaction between enzyme and substrate, and significantly improving the enzyme's catalytic efficiency and stability.

[0004] Patent application CN119506263A proposes a polymer with high-temperature and pressure resistance. This polymer, when combined with an enzyme, catalyzes the epoxidation of olefins to efficiently and continuously synthesize epoxides. Patent application CN118792287A proposes a method for preparing a polymer-enzyme complex, significantly improving the stability and biocompatibility of papain. The preparation process is simple and efficient. However, research on multi-enzyme encapsulation using block polymers is limited. (3) Summary of the invention

[0005] The present invention aims to provide a block polymer-based micellar multienzyme complex, preparation method, and application. The invention uses cyclodextrin-b-polyethylene glycol-b-polystyrene block polymer (CD-b-PEG-b-PS) to encapsulate multienzyme molecules to construct nanomicelles. The polymer-encapsulated multienzymes form a nanoconfined catalytic system, significantly improving the efficiency and stability of the multienzyme cascade catalysis. The micellar multienzyme complex, used as a catalyst, catalyzes the synthesis of D-psicose from maltodextrin, with significantly superior catalytic performance compared to the free multienzyme catalytic system, which is of great significance for promoting the industrial application of multienzyme biocatalysis technology.

[0006] The technical solution adopted in the present invention is:

[0007] The present invention provides a method for preparing a micellar multi-enzyme complex based on a block polymer, the method comprising the following steps:

[0008] (1) Dissolve chain transfer agent (CTA)-modified β-cyclodextrin (CD), azobisisobutyronitrile (AIBN), and styrene in dimethylformamide (DMF) and heat in an oil bath at 50-100°C under nitrogen for 1-3 days (preferably 75°C for 2 days);

[0009] (2) Dissolve polyethylene glycol monomethyl ether acrylate and AIBN in DMF, remove oxygen through nitrogen, and then add to the reaction solution of step (1), continue to react at 50-100°C for 1-3 days (preferably 75°C for 2 days), concentrate to remove the solvent, add water and allow to precipitate; dissolve the precipitate in DMF and add water and allow to precipitate, and vacuum dry the precipitate to obtain a yellow solid block polymer: cyclodextrin-b-polyethylene glycol-b-polystyrene (CD-b-PEG-b-PS, abbreviated as CPP);

[0010] (3) dissolving the block polymer in step (2) with a cosolvent to obtain a micellar solution;

[0011] (4) The micellar solution of step (3) was added to the mixed enzyme solution of starch phosphorylase (αGP), glucose phosphomutase (PGM), glucose phosphomutase (PGI), D-psicose-6-phosphate epimerase (A6PE) and D-psicose-6-phosphate phosphatase (A6PP), and the mixture was stirred and mixed to obtain the block polymer-based micellar multi-enzyme complex, which was recorded as αGP-PGM-PGI-A6PE-A6PP@CPP.

[0012] Furthermore, in step (1), the mass ratio of the chain transfer agent-modified β-cyclodextrin to azobisisobutyronitrile is 1:0.01-0.1 (preferably 1:0.036); the volume amount of styrene is 2-6 mL / g (preferably 4.4 mL / g) based on the mass of the chain transfer agent-modified β-cyclodextrin; and the volume amount of dimethylformamide is 20-50 mL / g (preferably 35 mL / g) based on the mass of the chain transfer agent-modified β-cyclodextrin.

[0013] Furthermore, in step (2), the mass ratio of polyethylene glycol monomethyl ether acrylate to azobisisobutyronitrile is 1:0.1-1 (preferably 1:0.1); and the volume amount of dimethylformamide added to the reaction solution of step (1) is 10-50 mL / g (preferably 36 mL / g) based on the mass of polyethylene glycol monomethyl ether acrylate.

[0014] Furthermore, the cosolvent in step (3) includes dimethyl sulfoxide (DMSO), DMF, ethyl acetate, petroleum ether, preferably DMSO.

[0015] Furthermore, in step (4), starch phosphorylase (αGP), phosphoglucomutase (PGM), phosphogluconate isomerase (PGI), D-psicose-6-phosphate epimerase (A6PE), and D-psicose-6-phosphate phosphatase (A6PP) are added in the form of pure enzyme solutions extracted from wet cells ultrasonically crushed and obtained by induction culture of recombinant genetically engineered bacteria containing the genes encoding the respective enzymes; the starch phosphorylase is derived from Thermotoga maritima MSB8 (Uniprot No. O33831), the phosphogluconate mutase is derived from the extremely thermophilic archaeon (Thermococcus kodakarensis, Uniprot No. Q68BJ6), and the phosphogluconate isomerase is derived from Thermus thermophilus HB8, Uniprot No. Q5SLL6), psicose 6-phosphate phosphatase is derived from Clostridium thermocellu (Uniprot No. A3DC21), and psicose 6-phosphate 3-epimerase is derived from Thermoanaerobacterium thermosaccharolyticum DSM 571 (Uniprot No. D9TQJ4), which have been disclosed in patent application CN114790469A.

[0016] Furthermore, pure enzyme solutions of starch phosphorylase, phosphoglucomutase, phosphoglucomutase, D-psicose-6-phosphate epimerase, and D-psicose-6-phosphate phosphatase are added to the mixed enzyme solution in an amount of 1-5 U / mL based on enzyme activity, and more preferably 2 U / mL, 3 U / mL, 2 U / mL, 4 U / mL, and 2 U / mL, respectively; and the amount of the micelle solution added is 0.05-1.5 mg / mL, preferably 0.1 mg / mL, based on the final concentration of the block polymer.

[0017] The present invention also provides a micellar multi-enzyme complex prepared by the method.

[0018] The present invention provides an application of the micellar multi-enzyme complex in catalyzing the synthesis of D-psicose from maltodextrin. The application method comprises the following steps: using the micellar multi-enzyme complex as a catalyst, maltodextrin as a substrate, MgCl2 and phosphate as auxiliary agents, and a pH 6 buffer as a reaction medium to form a reaction system, reacting at 50°C for 36 hours to obtain D-psicose.

[0019] Furthermore, in the reaction system, the catalyst is added to a final concentration of 50-70% (preferably 65%) by volume; maltodextrin is added to a final concentration of 5-15 g / L (preferably 10 g / L); MgCl2 is added to a final concentration of 1-10 mM (preferably 5 mM); and phosphate is added to a final concentration of 10-30 mM (preferably 20 mM).

[0020] Furthermore, the buffer solution is 50 mM Bis-Tris buffer solution (pH 6.0).

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] The block polymer-based micellar multi-enzyme complex provided by the present invention exhibits nanometer-confined space, increasing the substrate concentration within the relative space, significantly improving catalytic efficiency, and thus increasing yield. Using the micellar multi-enzyme complex as a catalyst for synthesizing D-psicose using inexpensive maltodextrin as a raw material not only saves costs but also enables efficient catalytic synthesis of D-psicose with a yield exceeding 70%. (IV) Description of the accompanying drawings

[0023] Figure 1 This is a transmission scanning electron microscopy image of the micelle-enzyme complex A6PP&A6PE@CPP.

[0024] Figure 2 Relative enzyme activities of A6PE&A6PP@CPP after encapsulation at different micelle concentrations.

[0025] Figure 3 Comparison of the reaction process of D-psicose synthesis from maltodextrin catalyzed by free multienzyme and micelle-enzyme complex αGP-PGM-PGI-A6PE-A6PP@CPP. (V) Specific implementation methods

[0026] The present invention is further described below with reference to specific examples, but the scope of protection of the present invention is not limited thereto: The CTA-functionalized β-cyclodextrin used in the examples of the present invention was prepared with reference to Ren L., Liu S., Sun Z.*, Harboring Organocatalysts in a Star-Shaped Block Copolymer for Micellar Catalysis and Emulsion Catalysis. Catal. Sci. Technol., 2024, 14, 255-260.

[0027] Example 1: Preparation of CPP

[0028] (1) CTA-functionalized β-cyclodextrin (900 mg, 0.35 mmol), azobisisobutyronitrile (AIBN, 32.5 mg), and 4 mL of styrene were dissolved in 30 mL of dimethylformamide (DMF). Nitrogen was then introduced for 30 min to remove oxygen, and the mixture was heated in an oil bath at 75 °C for two days.

[0029] (2) Polyethylene glycol monomethyl ether acrylate (molecular weight M n =480) (830 mg, 1.66 mmol) and AIBN (115 mg) were dissolved in 30 mL of DMF and nitrogen was passed through for 30 minutes before adding the reaction system of step (1). After continuing the reaction at 75°C for two days, the reaction solution was concentrated under reduced pressure until no liquid flowed out to remove the solvent. The concentrate was added with 3 times the volume of water and allowed to settle. The concentrate was centrifuged. The precipitate was dissolved in 3 times the volume of DMF and 3 times the volume of water was added to settle. The precipitate was centrifuged and the addition of DMF and water was repeated twice. The final precipitate was dried under vacuum at 55°C until no liquid flowed out to obtain 3.73 g of a yellow solid product, which was the block polymer: cyclodextrin-b-polyethylene glycol-b-polystyrene (CD-b-PEG-b-PS).

[0030] Example 2: Enzyme expression and protein purification

[0031] 1. Construction of recombinant bacteria

[0032] Starch phosphorylase from Thermotoga maritima MSB8 (Uniprot No. O33831), phosphoglucomutase from Thermococcus kodakarensis (Uniprot No. Q68BJ6), phosphoglucose isomerase from Thermus thermophilus HB8 (Uniprot No. Q5SLL6), psicose 6-phosphate phosphatase from Clostridium thermocellu (Uniprot No. A3DC21), and psicose 6-phosphate 3-epimerase (TtA6PE, Uniprot No. D9TQJ4) from Thermoanaerobacterium thermosaccharolyticum DSM 571 were synthesized by gene synthesis using the method of Example 2 of patent application CN114790469A. Simple Cloning (You C, Zhang XZ, Zhang The above genes were cloned into the pET20b vector (Novagen, Madison, WI) using the method described in [Y-HP. 2012. Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis. Appl. Environ. Microbiol. 78(5):1593-5] to obtain the corresponding expression vectors pET20b-TmαGP, pET20b-TkPGM, pET20b-TtcPGI, pET20b-CtA6PP, and pET20b-TmA6PE, respectively. The recombinant proteins were expressed in Escherichia coli BL21(DE3) to construct recombinant bacteria.

[0033] 2. Expression of recombinant bacteria

[0034] The recombinant strain obtained above was inoculated into LB liquid medium containing 50 μM kanamycin and cultured in a shaker at 37°C and 150 rpm for 12 h. Then, the inoculum was inoculated into 50 μM LB liquid medium containing kanamycin resistance at a volume concentration of 2% and cultured in a shaker at 37°C and 150 rpm until the bacterial concentration OD 600 To 0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1mM, induce culture at 28℃ for 12h, and centrifuge at 4℃ and 8000rpm for 10min to collect wet cells.

[0035] The cells obtained after centrifugation of the fermentation broth were resuspended in 50 mM Bis-Tris buffer solution at pH 7.0 at a volume of 30 g / L, and ultrasonically disrupted at 250 W for 20 min in an ice bath, with a disruption interval of 3 s for 1.5 s, and centrifuged (4°C, 12000 rpm, 10 min), and the supernatant was collected.

[0036] 3. Preparation of pure enzyme solution

[0037] (1) Thermal purification of α-GP, PGM, and PGI: 20 mL of the supernatant of α-GP, PGM, and PGI was placed in a 70°C water bath and incubated for 20 minutes to promote the thermal precipitation reaction. As the temperature rises, most of the impurities in the buffer solution undergo thermal denaturation and precipitate, while the target protein (thermophilic enzyme) continues to dissolve in the supernatant. Next, the mixture was centrifuged at 8,000 rpm for 20 minutes at 4°C, and the supernatant was collected to obtain 20 mL of pure enzyme solution of α-GP, PGM, and PGI, respectively.

[0038] (2) Pure D-psicose-6-phosphate epimerase (A6PE)

[0039] 20 mL of the supernatant was purified by a cobalt ion affinity chromatography column (purchased from Solebol, product model YA2420) according to the instructions. The eluate was collected and dialyzed against 50 mM Bis-Tris buffer solution, pH 6.0, at 4°C for 24 h. The intercepted liquid was collected to obtain 10 mL of pure A6PE enzyme solution.

[0040] (3) Pure D-psicose-6-phosphate phosphatase (A6PP)

[0041] 20 mL of the supernatant was purified by nickel affinity chromatography column (purchased from GenScript, product model L00684) according to the instructions. The eluate was collected and dialyzed against 50 mM Bis-Tris buffer solution, pH 6.0, at 4°C for 24 h. The intercepted liquid was collected to obtain 10 mL of pure A6PP enzyme solution.

[0042] Example 3: Preparation of micelle-enzyme complex (A6PE & A6PP@CPP) and enzyme activity detection

[0043] 1. Preparation of A6PE&A6PP@CPP

[0044] The block polymer CPP obtained in Example 1 was dissolved in DMSO to prepare a 20 mg / mL micelle solution. The A6PE pure enzyme solution and A6PP pure enzyme solution obtained in Example 2 were mixed at an enzyme activity ratio of 2:1 and added to the micelle solution. The amount of A6PE pure enzyme solution and A6PP pure enzyme solution added to the reaction solution was 4 U / mL and 2 U / mL respectively in terms of enzyme activity. The amount of micelle solution added was 0.1 mg / mL in terms of the final concentration of the block polymer CPP. The mixture was mixed at 15°C for 30 minutes to obtain a micelle-enzyme complex, which was recorded as A6PE&A6PP@CPP. The mixture was observed under a transmission electron microscope (TEM). The results were as follows: Figure 1 As shown, A6PP&A6PE@CPP can be successfully made into nanoscale size, thereby effectively improving the mass transfer function.

[0045] 2. Enzyme activity detection

[0046] Micellar multienzyme complex enzyme activity assay: 150 μL of A6PE&A6PP@CPP obtained in step 1 was added to 295 μL of 50 mM Bis-Tris buffer (pH 6.0). A final concentration of 5 mM MgCl2 and a final concentration of 10 mM fructose-6-phosphate (F6P) were then added. The mixture was reacted at 50°C for 5 min. The D-psicose content was determined by HPLC, and the activity of A6PE&A6PP@CPP was calculated.

[0047] Free enzyme activity assay: 100 μL of the pure A6PE enzyme solution and 50 μL of the pure A6PP enzyme solution obtained in Example 2 were added to 295 μL of 50 mM Bis-Tris buffer (pH 6.0). MgCl2 (final concentration: 5 mM) and fructose-6-phosphate (F6P) (final concentration: 10 mM) were then added. The mixture was reacted at 50°C for 5 min. The D-psicose content was determined by HPLC, and the activities of A6PE and A6PP were calculated.

[0048] Enzyme activity definition: the amount of enzyme required to produce 1 μmol of D-psicose per minute.

[0049] Relative enzyme activity (%) = enzyme activity of micelle-enzyme complex / enzyme activity of free A6PE & A6PP not embedded in micelles.

[0050] High performance chromatography detection conditions: Thermo Fisher liquid chromatograph U3000, differential refractive index detector, Bio-Rad HPX-87H column, detection at 60°C, 5 mM sulfuric acid as the mobile phase, and a flow rate of 0.6 mL / min.

[0051] Example 4: Activity determination of enzymes embedded in different micelle solution concentrations

[0052] The amount of micelle solution added in Example 3 was changed to 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.5 mg / mL, and 1 mg / mL respectively based on the final concentration of block polymer CPP. Other operations were the same. The relative enzyme activities of A6PE&A6PP@CPP prepared with micelle solutions of different concentrations were shown in Table 1. Figure 2 As shown, the control is free A6PE & A6PP.

[0053] The results showed that the optimal encapsulation effect was achieved when the micelle solution was added at a final concentration of 0.1 mg / mL of the block polymer CPP. Compared to the free enzyme, the relative enzymatic activity of the micelle-enzyme complex at this concentration reached 135.5%.

[0054] Example 5: Preparation of micelle-enzyme complex (αGP-PGM-PGI-A6PE-A6PP@CPP)

[0055] The block polymer CPP obtained in Example 1 was dissolved in DMSO to prepare a 20 mg / mL micelle solution.

[0056] The αGP pure enzyme solution, PGM pure enzyme solution, PGI pure enzyme solution, A6PE pure enzyme solution, and A6PP pure enzyme solution obtained in Example 2 were mixed in an enzyme activity ratio of 2:3:2:4:2 and added to the micelle solution. The amounts of αGP pure enzyme solution, PGM pure enzyme solution, PGI pure enzyme solution, A6PE pure enzyme solution, and A6PP pure enzyme solution added to the reaction solution were 2 U / mL, 3 U / mL, 2 U / mL, 4 U / mL, and 2 U / mL, respectively, based on enzyme activity. The amount of micelle solution added was 0.1 mg / mL based on the final concentration of the block polymer CPP. After mixing at 15° C. for 30 min, αGP-PGM-PGI-A6PE-A6PP@CPP was obtained.

[0057] Example 6: Synthesis of D-psicose by Micellar-Multienzyme Complex Cascade Catalysis

[0058] 1. Micellar-multienzyme complex as catalyst

[0059] The αGP-PGM-PGI-A6PE-A6PP@CPP obtained in Example 5 was used as a catalyst and maltodextrin was used as a substrate to produce D-psicose through a one-pot process.

[0060] To 10 mL of 50 mM Bis-Tris buffer (pH 6.0) was added 6.5 mL of catalyst, followed by a final concentration of 5 mM MgCl2, a final concentration of 20 mM phosphate, and a final concentration of 10 g / L maltodextrin. The reaction temperature was 50°C. Samples were taken every 2 hours to determine the yield of D-psicose using the method described in Example 3.

[0061] 2. Free polyenzymes as catalysts

[0062] The catalyst in step 1 was replaced with the αGP pure enzyme solution, PGM pure enzyme solution, PGI pure enzyme solution, A6PE pure enzyme solution, and A6PP pure enzyme solution prepared by the method of Example 1. The added amounts were 2 U / mL of α-GP, 3 U / mL of PGM, 2 U / mL of PGI, 4 U / mL of A6PE, and 2 U / mL of A6PP, respectively. The other operations were the same.

[0063] The results are as follows Figure 3 As shown, after 24 h of reaction, the yields of free enzyme and αGP-PGM-PGI-A6PE-A6PP@CPP reached 56% and 70%, respectively.

[0064] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a micellar multi-enzyme complex based on block polymers, characterized in that: The method comprises the following steps: (1) Dissolve chain transfer agent-modified β-cyclodextrin, azobisisobutyronitrile, and styrene in dimethylformamide, and heat in an oil bath at 50-100°C under nitrogen for 1-3 days; (2) dissolving polyethylene glycol monomethyl ether acrylate and azobisisobutyronitrile in dimethylformamide, removing oxygen through nitrogen, and then adding the mixture to the reaction solution of step (1), continuing the reaction at 50-100° C. for 1-3 days, concentrating to remove the solvent, adding water and allowing to stand for precipitation; dissolving the precipitate in dimethylformamide, adding water and allowing to stand for precipitation, and vacuum drying the precipitate to obtain a yellow solid block polymer; (3) dissolving the block polymer in step (2) with a cosolvent to obtain a micellar solution; (4) Adding the micellar solution of step (3) to the mixed enzyme solution of starch phosphorylase, glucose phosphomutase, glucose phosphomutase, D-psicose-6-phosphate epimerase, and D-psicose-6-phosphate phosphatase, stirring and mixing, thereby obtaining the block polymer-based micellar multi-enzyme complex.

2. The method according to claim 1, wherein In step (1), the mass ratio of the chain transfer agent-modified β-cyclodextrin to azobisisobutyronitrile is 1:0.01-0.1; the volume amount of styrene is 2-6 mL / g based on the mass of the chain transfer agent-modified β-cyclodextrin; and the volume amount of dimethylformamide is 20-50 mL / g based on the mass of the chain transfer agent-modified β-cyclodextrin.

3. The method according to claim 1, wherein In step (2), the mass ratio of polyethylene glycol monomethyl ether acrylate to azobisisobutyronitrile is 1:0.1-1; and the volume amount of dimethylformamide added to the reaction solution of step (1) is 10-50 mL / g based on the mass of polyethylene glycol monomethyl ether acrylate.

4. The method according to claim 1, wherein The cosolvent in step (3) includes dimethyl sulfoxide, dimethylformamide, ethyl acetate, and petroleum ether; and the concentration of the micelle solution is 0.05 to 1.5 mg / mL.

5. The method according to claim 1, wherein In step (4), starch phosphorylase, phosphoglucomutase, phosphoglucoisomerase, D-psicose-6-phosphate epimerase, and D-psicose-6-phosphate phosphatase are added in the form of pure enzyme solutions extracted from wet cells ultrasonically crushed and obtained by induction culture of recombinant genetically engineered bacteria containing the genes encoding the respective enzymes; the starch phosphorylase is derived from Thermotoga maritima MSB8 (Uniprot No. O33831), the phosphoglucomutase is derived from Thermococcus kodakarensis (Uniprot No. Q68BJ6), and the phosphoglucoisomerase is derived from Thermus thermophilus HB8, Uniprot No. Q5SLL6), psicose 6-phosphate phosphatase from Clostridium thermocellu (Uniprot No. A3DC21), and psicose 6-phosphate 3-epimerase from Thermoanaerobacterium thermosaccharolyticum DSM 571 (Uniprot No. D9TQJ4).

6. The method according to claim 5, wherein Pure enzyme solutions of starch phosphorylase, phosphoglucomutase, phosphoglucomutase, D-psicose-6-phosphate epimerase, and D-psicose-6-phosphate phosphatase are added to the mixed enzyme solution, and the added amount is 1-5 U / mL based on enzyme activity; the added amount of the micelle solution is 0.05-1.5 mg / mL based on the final concentration of the block polymer.

7. The method according to claim 6, wherein The amounts of pure enzyme solutions of starch phosphorylase, phosphoglucomutase, phosphoglucomutase, D-psicose-6-phosphate epimerase, and D-psicose-6-phosphate phosphatase added to the mixed enzyme solution were 2 U / mL, 3 U / mL, 2 U / mL, 4 U / mL, and 2 U / mL, respectively, based on enzyme activity.

8. A micellar multi-enzyme complex prepared by the method according to any one of claims 1 to 7.

9. Use of the micellar multi-enzyme complex according to claim 8 in catalyzing the synthesis of D-psicose from maltodextrin, the method comprising: using the micellar multi-enzyme complex as a catalyst, maltodextrin as a substrate, MgCl2 and phosphate as auxiliary agents, and a pH 6 buffer as a reaction medium to form a reaction system, reacting at 50°C for 36 hours to obtain D-psicose.

10. The use according to claim 9, characterized in that In the reaction system, the catalyst is added to a final concentration of 50-70% by volume; the maltodextrin is added to a final concentration of 5-15 g / L; the MgCl2 is added to a final concentration of 1-10 mM; and the phosphate is added to a final concentration of 10-30 mM.

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