Method for synthesizing D-psicose through multi-enzyme co-immobilization cascade combination

Abstract

The invention discloses a method for synthesizing D-psicose through multi-enzyme co-immobilization cascade combination, and belongs to the field of enzyme engineering. According to the method provided by the invention, D-psicose-3-epimerase, L-rhamnokinase, polyphosphate kinase and prepared nitrilotriacetic acid modified magnetic nanoparticles are mixed and incubated to obtain the co-immobilized enzyme. According to the efficient co-immobilized enzyme provided by the invention, the stability is improved, the efficient co-immobilized enzyme is easily separated from an enzymolysis system, the repeated utilization of the immobilized enzyme can be realized, and the production cost is reduced. When the co-immobilized enzyme is applied to preparation of D-psicose, the raw material cost of an enzyme preparation is greatly reduced, and the co-immobilized enzyme has relatively high industrial application value.

Description

Technical Field

[0001] The invention relates to a high-efficiency multi-enzyme co-immobilization cascade method and application thereof to the preparation of D-psicose, and belongs to the field of enzyme engineering. Background Art

[0002] D-psicose is the C-3 diastereomer of D-fructose and exists in the form of free sugar in wheat. In addition to its physiological functions such as low energy, improvement of intestinal flora, and anti-caries, D-psicose also plays an important physiological role in inhibiting blood sugar increase and body fat accumulation, scavenging free radicals, and neuroprotection. D-psicose has a sweetness of 70% of sucrose and only 10% of sucrose in energy. It is a new sweetener, especially suitable for people with diabetes and obesity. It can be absorbed by the small intestine and excreted from the body through urine. In 2011, D-psicose was recognized as a generally recognized as safe (GRAS) food by the US FDA, allowing it to be added to food. Since then, D-psicose has developed rapidly, and a variety of products containing D-psicose have appeared on the market. They are widely welcomed in the market as functional foods for special populations (such as diabetics and obese people).

[0003] There are two main methods for the synthesis of D-psicose: chemical synthesis and biotransformation. The chemical synthesis of D-psicose is relatively difficult. Molybdate is used as a catalyst and the reaction temperature is maintained at 80-120°C. There are many by-products in this process. At the same time, the chemical synthesis method also has problems such as serious environmental pollution and low stereoselectivity. Therefore, the synthesis of D-psicose mainly relies on biotransformation. The biotransformation method mainly uses enzyme catalytic reactions to produce D-psicose. Compared with the chemical synthesis method, the biotransformation method has the advantages of easy operation and no other by-products. There are two main biotransformation methods: (1) D-psicose is synthesized using dihydroxyacetone phosphate (DHAP)-dependent aldolase. DHAP can react with D / L-glyceraldehyde under the action of aldolase to produce phosphate sugars, which can then be dephosphorylated to various rare sugars such as D / L-psicose and D / L-tagatose by phosphatase; (2) D-psicose is synthesized using the Izumoring strategy. D-psicose is usually converted into D-psicose under the catalysis of D-psicose-3-epimerase using D-fructose as a substrate. However, the conversion of fructose to D-psicose catalyzed by D-psicose-3-epimerase is a reversible reaction with a maximum conversion rate of about 34%. Therefore, the generated D-psicose can be phosphorylated and then dephosphorylated to achieve the forward reaction, thereby improving the reaction efficiency.

[0004] The production of D-psicose by single enzyme method has problems such as low yield and difficulty in separation, and the enzyme is expensive. These have become the main bottlenecks restricting the efficient preparation of D-psicose. These problems can be effectively solved by multi-enzyme cascade immobilization technology. Multi-enzyme cascade reaction is a biocatalytic process that converts a substrate into a final product through the synergistic action of multiple enzymes. This reaction can achieve the controllability and efficiency of complex chemical transformations and has broad application prospects. In the multi-enzyme cascade reaction, multiple enzymes are linked together in a certain order, and are organically combined through various mechanisms such as enzyme substrates, enzyme-enzyme interactions, and auxiliary factors, thereby improving the yield and purity of the product and shortening the reaction time. Immobilized enzyme refers to the enzyme fixed in a certain space with the help of carrier materials for reaction, which can effectively resist the influence of environmental changes on enzyme activity, and is easy to recycle and use the enzyme, simplifying the subsequent separation and purification steps, and realizing the reuse of the enzyme. Therefore, the development of an efficient and practical multi-enzyme immobilization cascade to produce D-psicose can greatly reduce production costs.

[0005] A variety of methods are currently used to achieve cascade production of D-psicose. For example, Chinese patent CN117646046A discloses a method for using a dual-enzyme cascade to recombinant Bacillus subtilis to catalyze the biosynthesis of D-psicose from D-glucose, which successfully achieves dual-enzyme cascade synthesis of D-psicose, but its overall conversion rate is only 15%. Therefore, there is an urgent need to find a method for efficiently preparing a multi-enzyme immobilized cascade production of D-psicose with stable enzyme activity and multiple reuses. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for the co-immobilization and cascade synthesis of D-psicose by multiple enzymes, aiming to solve the technical problem that the prior art lacks a method for efficiently preparing a multi-enzyme immobilization cascade production of D-psicose with stable enzyme activity and repeatable reuse.

[0007] The first technical solution provided by the present invention is a method for preparing a novel immobilized carrier, wherein the method comprises covering the surface of Fe3O4 nanoparticles with a shell of zeolite imidazole framework (ZIF-8), then introducing aldehyde groups using 3-aminopropyltriethoxysilane and glutaraldehyde, and finally introducing NTA using Nα,Nα-bis(carboxymethyl)-L-lysine, thereby finally synthesizing NTA-functionalized magnetic nanoparticles, i.e., immobilized carriers.

[0008] In one embodiment, the method comprises the steps of:

[0009] (1) Preparation of magnetic nanoparticles coated with zeolite imidazole framework-8 (ZIF-8): Fe3O4 was dissolved in a polystyrene sulfonate (PSS) solution, and then ultrasonicated and stirred at room temperature. After 30 minutes, the nanoparticles were recovered with a magnet, washed with methanol, and then the magnetic nanoparticles were dispersed in methanol; then a soluble zinc salt was added to the solution and stirred for 10 minutes, and then 2-methylimidazole was slowly added to the solution and stirred for 4 hours. After the end, the magnet was recovered and washed with ethanol for 3 to 4 times, and then dried at low temperature;

[0010] (2) Aldehyde modification: Take an appropriate amount of the magnetic nanoparticles prepared in (1) and ultrasonically disperse them in ethanol, then add an appropriate amount of 3-aminopropyltriethoxysilane (APTES) and stir to react for 6 hours. After the reaction, wash with pure water several times and then disperse in an appropriate amount of pure water. Then, slowly add an appropriate amount of 50% glutaraldehyde while stirring and stir for 3 hours to obtain aldehyde-modified magnetic nanoparticles. After the reaction, the magnet is recovered and washed with pure water 3 to 4 times.

[0011] (3) Nitrilotriacetic acid (NTA) functionalization: The magnetic nanoparticles washed in (2) were ultrasonically dispersed in an appropriate amount of pure water, and an appropriate amount of 50 mM, pH 8 Nα, Nα-bis(carboxymethyl)-L-lysine hydrate solution was added and stirred for 24 h. After the reaction, the magnetic nanoparticles were washed with ultrapure water for 3 to 4 times. The washed magnetic nanoparticles were resuspended in a NiCl2 (50 mM) aqueous solution and stirred for 2 h, and then washed with pure water for 3 to 4 times. After low-temperature drying, the magnetic nanoparticles functionalized with nitrilotriacetic acid were obtained.

[0012] In one embodiment, in step (1), the particle size of the Fe3O4 is 200 nm, and the concentration of the sodium polystyrene sulfonate solution is 0.3-0.5% w / v.

[0013] In one embodiment, in step (1), the soluble zinc salt is Zn(NO3)2, ZnSO4 or ZnCl2.

[0014] In one embodiment, in step (1), the addition ratio of Fe3O4 and sodium polystyrene sulfonate solution is (3:2)-(2:1) (w / v), and the addition ratio of Fe3O4, methanol, Zn(NO3)2 and 2-methylimidazole is (1:1:5:14)-(2:1:5:14) (w / v / w / w).

[0015] In one embodiment, the temperature of the low-temperature drying is 40° C. to 50° C., and the time of the low-temperature drying is 12 hours.

[0016] In one embodiment, in step (2), the magnetic nanoparticles prepared in (1) are added to 3-aminopropyltriethoxysilane and ethanol in a ratio of (30:1:10)-(60:1:10) (w / v / v), and to pure water and 50% glutaraldehyde in a ratio of (10:1:1)-(20:1:1) (w / v / v).

[0017] In one embodiment, in step (3), the magnetic nanoparticles prepared in (2) are added to pure water and Nα, Nα-bis(carboxymethyl)-L-lysine hydrate solution in a ratio of (20:5:1)-(40:5:1) (w / v / v), and to the NiCl2 aqueous solution in a ratio of (4:1)-(6:1) (w / v).

[0018] The second technical solution provided by the present invention is an immobilized carrier prepared by the method described in the first technical solution.

[0019] The third technical solution provided by the present invention is a method for preparing a cascade immobilized enzyme, wherein the method comprises mixing and incubating D-psicose-3-epimerase, L-rhamnose kinase, and polyphosphate kinase with the immobilized carrier described in the second technical solution.

[0020] In one embodiment, D-psicose-3-epimerase with a histidine tag, L-rhamnokinase, polyphosphate kinase, and the immobilized carrier are mixed in a mass ratio of (1:1:1:50) to (3:1:1:50).

[0021] In one embodiment, the incubation time is 15 to 120 minutes.

[0022] In one embodiment, the D-psicose-3-epimerase is derived from Clostridium cellulolyticum H10, the L-rhamnokinase is derived from Escherichia coli, and the polyphosphate kinase is derived from Sinorhizobium meliloti.

[0023] The fourth technical solution provided by the present invention is a method for preparing a cascade immobilized enzyme using the method described in the third technical solution.

[0024] The fifth technical solution provided by the present invention is a method for preparing D-psicose, wherein D-fructose is used as a substrate, the cascade immobilized enzyme described in the fourth technical solution is used as a catalyst to catalyze the generation of D-psicose-1-phosphate, and then D-psicose is obtained by dephosphorylation by acid phosphatase.

[0025] In one embodiment, the added mass ratio of D-fructose and the co-immobilized enzyme is 4:1.

[0026] In one embodiment, the D-fructose concentration is 5-40 mg / mL and the co-immobilized enzyme concentration is 4 mg / mL.

[0027] In one embodiment, the catalytic reaction conditions are as follows: pH 8, 30-40°C and 5 mM Mn 2+ The reaction was continued for 2-12 hours.

[0028] The sixth technical solution provided by the present invention is the application of the method described in the first technical solution, or the immobilized carrier described in the second technical solution, or the method described in the third technical solution, or the cascade immobilized enzyme described in the fourth technical solution, or the method described in the fifth technical solution in the fields of medicine, food and daily necessities.

[0029] The seventh technical solution provided by the present invention is the use of the method described in the first technical solution, or the immobilized carrier described in the second technical solution, or the method described in the third technical solution, or the cascade immobilized enzyme described in the fourth technical solution, or the method described in the fifth technical solution in the preparation of a product containing D-psicose.

[0030] The technical effects of the present invention are as follows:

[0031] 1. The three-enzyme co-immobilization cascade method provided by the present invention uses magnetic nanoparticles coated with zeolite imidazole skeletons as carriers, connects the three enzymes on the surface of magnetic nanoparticles in proportion through affinity binding, and the conversion rate of the obtained co-immobilized enzymes can reach up to more than 78%.

[0032] 2. The cascade co-immobilized enzyme prepared by the present invention has good recycling performance. After five cycles, the conversion rate is about 36.8%, and the relative enzyme activity retention rate is about 57.3%.

[0033] 3. The conversion rate of the cascade co-immobilized enzyme reaction prepared by the present invention for 12 hours was increased from about 31% to about 72% compared with that of the immobilized single enzyme, which was a significant increase.

[0034] In summary, the present invention adopts the principle of affinity binding of histidine tag and nitrilotriacetic acid for the first time to co-immobilize D-psicose-3-epimerase, L-rhamnose kinase and polyphosphate kinase, which can effectively improve the stability of the enzymes and realize repeated use. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The changes of the immobilization rates of the three enzymes over time.

[0036] Figure 2The conversion rate of D-psicose synthesized by immobilized three-enzyme cascade under the conditions.

[0037] Figure 3 Diagram for cascade co-immobilized enzyme recycling.

[0038] Figure 4 This is a graph showing the conversion rate of D-psicose under different enzyme ratio cascades.

[0039] Figure 5 The D-psicose conversion rate of immobilized single enzyme and cascade immobilized enzyme. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0041] Test method:

[0042] 1. Determination of enzyme activity of cascade co-immobilized enzyme: After the reaction is completed, the immobilized enzyme is recovered by a magnet, and then the supernatant is transferred to a boiling water bath for 5 minutes. After cooling, it is centrifuged at 10,000 rpm for 1 minute, and then filtered through a 0.22 μm filter membrane. The reduction of D-fructose is detected by high performance liquid chromatography. One enzyme activity unit (U) is defined as the reaction of 1 μmol D-fructose per mg enzyme per minute.

[0043] 2. Determination of the activity of immobilized L-rhamnose kinase: After the reaction, the immobilized enzyme was recovered by magnet, and then the supernatant was transferred to a boiling water bath for 5 minutes. After cooling, it was centrifuged at 10,000 rpm for 1 minute, and then filtered through a 0.22 μm filter membrane. The reduction of D-psicose was detected by high performance liquid chromatography. One enzyme activity unit (U) was defined as the reaction of 1 μmol D-psicose per mg enzyme per minute.

[0044] 3. Determination of the activity of immobilized polyphosphate kinase: After the reaction is completed, the immobilized enzyme is recovered by a magnet, and then the supernatant is transferred to a boiling water bath for 5 minutes. After cooling, it is centrifuged at 10,000 rpm for 1 minute, and then filtered through a 0.22 μm filter membrane. The amount of ATP generated is detected by high-performance liquid chromatography. One unit of enzyme activity (U) is defined as 1 μmol ATP generated per mg enzyme per minute.

[0045] 4. HPLC detection method for the activity of co-immobilized enzyme and immobilized L-rhamnose sugar kinase: Waters HPLC, e2414 differential detector, COSMOSIL Sugar-D 4.6 mm × 250 mm chromatographic column; mobile phase: 80% acetonitrile, flow rate: 1.0 ml / min, column temperature: 30°C.

[0046] 5. HPLC detection method for immobilized polyphosphate kinase activity: Waters HPLC, UV detector, Xbridge C18 4.6 mm × 250 mm chromatographic column; mobile phase: 0.1 mM pH 6 sodium phosphate buffer, flow rate: 1.0 ml / min, column temperature: 30°C.

[0047] The raw materials used in the examples are:

[0048] 1. E. coli BL21(DE3) / pET22b-dpe has been published in the reference: Mu W, Chu F, Xing Q, et al. Cloning, Expression, and Characterization of a D-Psicose 3-Epimerase from Clostridium cellulolyticum H10[J]. J Agric Food Chem, 2011, 59(14): 7785-7792.

[0049] 2. Plasmid pET28a-rhabRhab: Use 6×His-tag as the short peptide fused to the C-terminus of the protein, insert Rhab (genebank: CAD6021149.1) between the BmHⅠ and XhoⅠ restriction sites of plasmid pET28a, and construct the recombinant plasmid pET28a-rhab. Transform the recombinant plasmid pET28a-rhab into E. coli BL21 (DE3) to construct the recombinant strain E. coli BL21 (DE3) / pET28a-rhab.

[0050] 3. Plasmid pET28a-ppk was published in: Feng Linxue. Study on the synthesis of D-psicose from D-fructose catalyzed by multiple enzymes [D]. Jiangnan University, 2021. The plasmid pET28a-ppk was transformed into Escherichia coli E. coli BL21 (DE3) to construct the recombinant strain E. coli BL21 (DE3) / pET28a-ppk.

[0051] 4. LB liquid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L;

[0052] LB solid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L;

[0053] The culture medium was prepared with deionized water and sterilized at 121°C for 20 min.

[0054] 5. Nα, Nα-bis(carboxymethyl)-L-lysine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Acid phosphatase was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0055] Example 1: Preparation of cascade immobilized enzyme

[0056] The specific steps are as follows:

[0057] (1) Enzyme solution preparation: E. coli BL21(DE3) / pET22b-dpe, E. coli BL21(DE3) / pET28a-rhab and E. coli BL21(DE3) / pET28a-ppk strains were inoculated into LB medium, cultured in a constant temperature shaker at 37°C for about 2 hours, and then IPTG with a final concentration of 1 mM was added to induce expression, and the cells were collected by centrifugation. The cells were resuspended in buffer, disrupted with an ultrasonic disruptor, and the supernatant was collected by centrifugation to obtain a crude enzyme solution; His Trap HP 1mL Ni 2+ The crude enzyme solution was purified by affinity chromatography to obtain three enzymes.

[0058] (2) Preparation of carrier: 1. Preparation of magnetic nanoparticles coated with zeolite imidazole framework-8 (ZIF-8): 300 mg Fe3O4 (200 nm) was dissolved in 200 mL polystyrene sulfonate (PSS) solution (0.3%, w / v), then ultrasonicated and stirred at room temperature, and after 30 min, the nanoparticles were recovered with a magnet, washed with methanol, and then the magnetic nanoparticles were dispersed in 300 mL methanol; then 1.5 g Zn(NO3)2 was added to the solution and stirred for 10 min, and then 4.2 g 2-methylimidazole was slowly added to the solution and stirred for 4 h. After the end, the magnet was recovered and washed with ethanol for 3 to 4 times and then dried at 40°C to 50°C for 12 h; 2. Aldehyde modification: The magnetic nanoparticles prepared in the first step were ultrasonically dispersed in 40 mL ethanol, and then 6 mL 3-aminopropyltriethoxysilane (APTES), and stir for 6 hours; after the reaction, wash with pure water several times and disperse in 30mL pure water, slowly add 30mL 50% glutaraldehyde while stirring, stir for 3 hours to obtain aldehyde-modified magnetic nanoparticles, recover the magnet after the end, and wash with pure water 3 to 4 times. 3. Nitrilotriacetic acid (NTA) functionalization: ultrasonically disperse the magnetic nanoparticles washed in the second step in 60mL pure water, add 12mL 50mM, pH8 (50mM PBS) Nα, Nα-bis (carboxymethyl) -L-lysine hydrate solution, and stir for 24 hours. After the reaction, wash with ultrapure water 3 to 4 times. The washed magnetic nanoparticles are resuspended in 60mL NiCl2 (50mM) aqueous solution and stirred for 2 hours. After the reaction, wash with pure water 3 to 4 times, and dry at low temperature to obtain nitrilotriacetic acid functionalized magnetic nanoparticles.

[0059] (3) Three-enzyme cascade co-immobilization:

[0060] The purified DPE, RHAB, and PPK were ultrafiltered to remove imidazole, and the ultrafiltration cut-off molecular weight was 10KDa. Then the enzyme concentration was adjusted to 1mg / mL, and the immobilized carrier concentration was adjusted to 50mg / mL. 1ml of enzyme solution was mixed with 1ml of carrier, and fixed at 16°C for 15-120min. Then the supernatant was removed, washed with deionized water 3 times, and finally resuspended with 2ml of deionized water.

[0061] Example 2: Preparation of cascade immobilized enzyme

[0062] The specific steps are as follows:

[0063] (1) Enzyme solution preparation: E. coli BL21(DE3) / pET22b-dpe, E. coli BL21(DE3) / pET28a-rhab and E. coli BL21(DE3) / pET28a-ppk strains were inoculated into LB medium, cultured in a constant temperature shaker at 37°C for about 2 hours, and then IPTG with a final concentration of 1 mM was added to induce expression, and the cells were collected by centrifugation. The cells were resuspended in buffer, disrupted with an ultrasonic disruptor, and the supernatant was collected by centrifugation to obtain a crude enzyme solution; His Trap HP 1mL Ni 2+ The crude enzyme solution was purified by affinity chromatography to obtain three enzymes.

[0064] (2) Preparation of carrier: 1. Preparation of magnetic nanoparticles coated with zeolite imidazole framework-8 (ZIF-8): 300 mg Fe3O4 (200 nm) was dissolved in 200 mL polystyrene sulfonate (PSS) solution (0.3%, w / v), then ultrasonicated and stirred at room temperature, and after 30 min, the nanoparticles were recovered with a magnet, washed with methanol, and then the magnetic nanoparticles were dispersed in 300 mL methanol; then 1.5 g Zn(NO3)2 was added to the solution and stirred for 10 min, and then 4.2 g 2-methylimidazole was slowly added to the solution and stirred for 4 h. After the end, the magnet was recovered and washed with ethanol for 3 to 4 times and then dried at 40°C to 50°C for 12 h; 2. Aldehyde modification: The magnetic nanoparticles prepared in the first step were ultrasonically dispersed in 40 mL ethanol, and then 6 mL 3-aminopropyltriethoxysilane (APTES), stirred for 6 hours; after the reaction, washed with pure water several times and dispersed in 30mL pure water, slowly added 30mL 50% glutaraldehyde while stirring, stirred for 3 hours to obtain aldehyde-modified magnetic nanoparticles, after the end, the magnet was recovered, and washed with pure water 3 to 4 times. 3. Nitrilotriacetic acid (NTA) functionalization: ultrasonically disperse the magnetic nanoparticles washed in the second step in 60mL pure water, add 12mL 50mM, pH8 (50mM PBS) Nα, Nα-bis (carboxymethyl) -L-lysine hydrate solution, stir for 24 hours, after the reaction, wash with ultrapure water 3 to 4 times. Resuspend the washed magnetic nanoparticles in 60mL NiCl2 (50mM) aqueous solution and stir for 2 hours. After the reaction, wash with pure water 3 to 4 times, and dry at low temperature to obtain nitrilotriacetic acid functionalized magnetic nanoparticles.

[0065] (3) Three-enzyme cascade co-immobilization:

[0066] The purified DPE, RHAB, and PPK were ultrafiltered to remove imidazole, and the ultrafiltration cut-off molecular weight was 10KDa. Then the enzyme concentration was adjusted to 1mg / mL, and the immobilized carrier concentration was adjusted to 50mg / mL. 2ml, 1ml, and 1ml of the enzyme solution of the three enzymes were mixed with 1ml of the carrier in sequence, and fixed at 16°C for 15 to 120min. Then the supernatant was removed, washed with deionized water 3 times, and finally resuspended with 2ml of deionized water.

[0067] Example 3: Preparation of cascade immobilized enzyme

[0068] The specific steps are as follows:

[0069] (1) Enzyme solution preparation: E. coli BL21(DE3) / pET22b-dpe, E. coli BL21(DE3) / pET28a-rhab and E. coli BL21(DE3) / pET28a-ppk strains were inoculated into LB medium, cultured in a constant temperature shaker at 37°C for about 2 hours, and then IPTG with a final concentration of 1 mM was added to induce expression, and the cells were collected by centrifugation. The cells were resuspended in buffer, disrupted with an ultrasonic disruptor, and the supernatant was collected by centrifugation to obtain a crude enzyme solution; His Trap HP 1mL Ni 2+ The crude enzyme solution was purified by affinity chromatography to obtain three enzymes.

[0070] (2) Preparation of carrier: 1. Preparation of magnetic nanoparticles coated with zeolite imidazole framework-8 (ZIF-8): 300 mg Fe3O4 (200 nm) was dissolved in 200 mL polystyrene sulfonate (PSS) solution (0.3%, w / v), then ultrasonicated and stirred at room temperature. After 30 min, the nanoparticles were recovered with a magnet, washed with methanol, and then the magnetic nanoparticles were dispersed in 300 mL methanol; 1.5 g Zn(NO3)2 was added to the solution while stirring and stirred for 10 min, and then 4.2 g 2-methylimidazole was slowly added to the solution and stirred for 4 h. After the end, the magnet was recovered and washed with ethanol for 3 to 4 times and then dried at 40°C to 50°C for 12 h; 2. Aldehyde modification: The magnetic nanoparticles prepared in the first step were ultrasonically dispersed in 40 mL ethanol, and then 6 mL 3-aminopropyltriethoxysilane (APTES), stirred for 6 hours; after the reaction, washed with pure water several times and dispersed in 30mL pure water, slowly added 30mL 50% glutaraldehyde while stirring, stirred for 3 hours to obtain aldehyde-modified magnetic nanoparticles, after the end, the magnet was recovered, and washed with pure water 3 to 4 times. 3. Nitrilotriacetic acid (NTA) functionalization: ultrasonically disperse the magnetic nanoparticles washed in the second step in 60mL pure water, add 12mL 50mM, pH8 (50mM PBS) Nα, Nα-bis (carboxymethyl) -L-lysine hydrate solution, stir for 24 hours, after the reaction, wash with ultrapure water 3 to 4 times. Resuspend the washed magnetic nanoparticles in 60mL NiCl2 (50mM) aqueous solution and stir for 2 hours. After the reaction, wash with pure water 3 to 4 times, and dry at low temperature to obtain nitrilotriacetic acid functionalized magnetic nanoparticles.

[0071] (3) Three-enzyme cascade co-immobilization:

[0072] The purified DPE, RHAB, and PPK were ultrafiltered to remove imidazole, and the ultrafiltration cut-off molecular weight was 10KDa. Then the enzyme concentration was adjusted to 1mg / mL, and the immobilized carrier concentration was adjusted to 50mg / mL. 3ml, 1ml, and 1ml of the enzyme solution of the three enzymes were mixed with 1ml of the carrier in sequence, and fixed at 16°C for 15 to 120min. Then the supernatant was removed, washed with deionized water 3 times, and finally resuspended with 2ml of deionized water.

[0073] Example 4 Three-enzyme cascade immobilization time

[0074] The purified DPE, RHAB, and PPK were ultrafiltered to remove imidazole, and the ultrafiltration cut-off molecular weight was 10KDa. Then the enzyme concentration was adjusted to 1mg / mL, and the immobilized carrier concentration was adjusted to 50mg / mL. 1ml of enzyme solution was taken from each of the three enzymes and mixed with 1ml of carrier, and fixed at 16°C for 15 to 120min. Then the supernatant was removed, washed with deionized water 3 times, and finally resuspended with 2ml of deionized water to obtain the respective immobilized single enzymes.

[0075] Depend on Figure 1 It can be seen that, with the 120-min carrier fixation quality as the control, the three enzymes can be fixed for 15 minutes to achieve a relative immobilization rate of 95%, and the immobilization efficiency is greatly improved compared with traditional adsorption and encapsulation.

[0076] Example 5 Reaction conditions of cascade immobilized enzyme

[0077] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8, 20 mM ATP, 10 mM Poly6), 50 μL of the cascade co-immobilized enzyme prepared in Examples 1, 2, and 3 were added, and the reaction was carried out for 2-12 hours respectively, and the conversion rate was measured.

[0078] 1. The impact of time on conversion rate:

[0079] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8, 20 mM ATP, 10 mM Poly6), 50 μL of the co-immobilized enzyme prepared in Examples 1, 2, and 3 were added, and the reaction was carried out for 2-12 hours respectively, and the conversion rate was measured.

[0080] 2. Effect of temperature on conversion rate:

[0081] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8, 20 mM ATP, 10 mM Poly6), 50 μL of the co-immobilized enzyme prepared in Examples 1, 2, and 3 were added, and the reaction was carried out at 30° C. and 35° C., respectively, and the conversion rate was measured.

[0082] from Figure 2 It can be seen that when the reaction temperature of the co-immobilized enzyme was 30°C and the reaction time was 12 h, the conversion rate reached a maximum of 72%.

[0083] Example 6 Reusability of Cascade Immobilized Enzymes

[0084] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8, 20 mM ATP, 10 mM Poly6), 50 μL of the co-immobilized enzyme prepared in Example 2 was added, and the mixture was reacted at 30° C. for 6 h and then recovered using a magnet. The above-mentioned conditions were then repeated for 5 cycles, and the conversion rate and relative enzyme activity retention rate of each cycle were measured.

[0085] Depend on Figure 3 It can be seen that after five cycles, the conversion rate was about 36.8% and the relative enzyme activity retention rate was about 57.3%.

[0086] Example 7 Preparation of D-psicose

[0087] (1) Take 200 μL of substrate solution (10 mg / mL fructose, 5 mM Mn 2+ , pH 8, 10 mM ATP, 5 mM Poly6), 50 μL of the cascaded co-immobilized enzyme prepared in Example 2 was added, and the reaction was carried out at 30°C for 2-16 h, then the immobilized enzyme was recovered by a magnet, the supernatant was adjusted to pH 5 with 0.1 mol / L hydrochloric acid solution, and then 1 μL of acid phosphatase was added to react for 12 h, and the conversion rate was measured.

[0088] (2) Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8, 20 mM ATP, 10 mM Poly6), 50 μL of the cascaded co-immobilized enzyme prepared in Example 2 was added, and the reaction was carried out at 30°C for 2-16 h, and then the immobilized enzyme was recovered by a magnet, the supernatant was adjusted to pH 5 with 0.1 mol / L hydrochloric acid solution, and then 1 μL of acid phosphatase was added to react for 12 h, and the conversion rate was measured.

[0089] (3) Take 200 μL of substrate solution (30 mg / mL fructose, 5 mM Mn 2+ , pH 8, 30 mM ATP, 15 mM Poly6), 50 μL of the cascaded co-immobilized enzyme prepared in Example 2 was added, and the reaction was carried out at 30°C for 2-16 h, then the immobilized enzyme was recovered by a magnet, the supernatant was adjusted to pH 5 with 0.1 mol / L hydrochloric acid solution, and then 1 μL of acid phosphatase was added to react for 12 h, and the conversion rate was measured.

[0090] Depend on Figure 4 It can be seen that under the condition (2), the conversion rate reached a maximum of about 78%.

[0091] Comparative Example 1: Immobilization of single enzyme and co-immobilization of three enzyme cascade

[0092] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8, 20 mM ATP, 10 mM Poly6), added 50 μL of immobilized D-psicose-3-epimerase and cascaded co-immobilized enzyme prepared in Example 2, reacted at 30° C. for 0.25-12 h, and measured the conversion rate.

[0093] Depend on Figure 5 It can be seen that the conversion rate of immobilized D-psicose-3-epimerase did not change significantly after reaching 31% after 1 h of reaction, while the conversion rate of the cascade immobilized enzyme finally reached 73.2% after 12 h of reaction, which was significantly improved compared with the immobilized single enzyme.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a cascade immobilized enzyme, characterized in that: The method comprises the following steps of mixing and incubating D-psicose-3-epimerase, L-rhamnose kinase and polyphosphate kinase with a histidine tag and an immobilized carrier; the preparation method of the immobilized carrier comprises the following steps of covering a layer of a zeolite imidazole skeleton shell on the surface of a Fe3O4 nanoparticle, then introducing an aldehyde group using 3-aminopropyltriethoxysilane and glutaraldehyde, and finally introducing NTA using Nα,Nα-bis(carboxymethyl)-L-lysine, and finally synthesizing an NTA-functionalized magnetic nanoparticle, namely the immobilized carrier.

2. The method according to claim 1, characterized in that The steps include: (1) Preparation of magnetic nanoparticles coated with zeolite imidazole framework-8 (ZIF-8): Fe3O4 was dissolved in a sodium polystyrene sulfonate solution, and then the nanoparticles were recovered by a magnet after being ultrasonicated and stirred at room temperature, and then washed with methanol, and then the magnetic nanoparticles were dispersed in methanol; a soluble zinc salt was added to the solution and stirred, and then 2-methylimidazole was slowly added to the solution and stirred, and then the magnet was recovered, washed with ethanol, and then dried at low temperature; (2) Aldehyde modification: The magnetic nanoparticles prepared in (1) are ultrasonically dispersed in ethanol, and then 3-aminopropyltriethoxysilane is added and stirred. After the reaction is completed, the nanoparticles are washed with pure water for several times and then dispersed in an appropriate amount of pure water. 50% glutaraldehyde is slowly added while stirring to obtain aldehyde-modified magnetic nanoparticles. After the reaction is completed, the magnetic nanoparticles are recovered and washed with pure water. (3) Nitrilotriacetic acid functionalization: ultrasonically disperse the washed magnetic nanoparticles in (2) in pure water, add 50 mM, pH 8 Nα, Nα-bis(carboxymethyl)-L-lysine hydrate solution and stir. After the reaction, wash with ultrapure water, resuspend the magnetic nanoparticles in 50 mM NiCl2 aqueous solution and stir. After the reaction, wash with pure water and dry at low temperature to obtain nitrilotriacetic acid functionalized magnetic nanoparticles, which are immobilized carriers. (4) Enzyme immobilization: D-psicose-3-epimerase, L-rhamnose kinase, and polyphosphate kinase with histidine tags are mixed and incubated with the immobilized carrier in step (3).

3. The method according to claim 2, characterized in that In step (1), the particle size of the Fe3O4 is 200nm, and the concentration of the sodium polystyrene sulfonate solution is 0.3-0.5% w / v; the addition ratio of the Fe3O4 and the sodium polystyrene sulfonate solution is (3:2)-(2:1) (w / v), and the addition ratio of Fe3O4, methanol, Zn(NO3)2 and 2-methylimidazole is (1:1:5:14)-(2:1:5:14) (w / v / w / w).

4. The method according to claim 2, characterized in that: In step (2), the magnetic nanoparticles prepared in (1) are added to 3-aminopropyltriethoxysilane and ethanol in a ratio of (30:1:10)-(60:1:10) (w / v / v), and to pure water and 50% glutaraldehyde in a ratio of (10:1:1)-(20:1:1) (w / v / v).

5. The method according to claim 2, characterized in that: In step (3), the magnetic nanoparticles prepared in (2) are added to pure water and Nα,Nα-bis(carboxymethyl)-L-lysine hydrate solution in a ratio of (20:5:1)-(40:5:1) (w / v / v), and to NiCl2 aqueous solution in a ratio of (4:1)-(6:1) (w / v).

6. The method according to claim 2, characterized in that The temperature of the low-temperature drying is 40° C. to 50° C., and the time of the low-temperature drying is 12 hours.

7. The method according to claim 2, characterized in that: In step (4), D-psicose-3-epimerase with a histidine tag, L-rhamnose kinase, polyphosphate kinase and an immobilized carrier are mixed in a mass ratio of (1:1:1:50) to (3:1:1:50); and the incubation time is 15 to 120 minutes.

8. A cascade immobilized enzyme prepared by the method according to any one of claims 1 to 7.

9. A method for preparing D-psicose, characterized in that: The method uses D-fructose as a substrate, utilizes the cascade immobilized enzyme described in claim 8 as a catalyst, catalyzes the generation of D-psicose-1-phosphate, and then dephosphorylates it through acid phosphatase to obtain D-psicose.

10. Use of the method according to any one of claims 1 to 7, or the cascade immobilized enzyme according to claim 8, or the method according to claim 9 in the preparation of a product containing D-psicose.

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