Method for efficiently immobilizing enzyme and application of enzyme in preparation of D-psicose

Abstract

The invention discloses a method for efficiently immobilizing enzyme and application of the method to preparation of D-psicose, and belongs to the field of enzyme engineering. According to the method provided by the invention, the immobilized D-psicose-3-epimerase is obtained by mixing and incubating the D-psicose-3-epimerase and prepared magnetic nanoparticles modified by nitrilotriacetic acid. According to the efficient immobilized D-psicose-3-epimerase provided by the invention, not only can the thermal stability of the D-psicose-3-epimerase be improved, but also the D-psicose-3-epimerase can be easily separated from an enzymolysis system, so that the reuse of the D-psicose-3-epimerase is realized, and the production cost is reduced. When the immobilized D-psicose-3-epimerase is applied to preparation of D-psicose, the raw material cost of an enzyme preparation is greatly reduced, and the immobilized D-psicose-3-epimerase has a relatively high industrial application value.

Description

Technical Field

[0001] The invention relates to a method for efficiently fixing an enzyme and application of the method 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 usually uses D-fructose as a substrate, and D-fructose is converted into D-psicose under the catalysis of D-psicose-3-epimerase.

[0004] However, the enzymatic production of D-psicose 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. Enzyme immobilization technology can effectively solve these problems. 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 immobilized D-psicose-3-epimerase can greatly reduce production costs.

[0005] A variety of methods are currently used to immobilize D-psicose-3-epimerase. For example, Chinese patent CN110016472B discloses a DTE immobilized nano-microsphere and a preparation method thereof and a method for producing D-psicose based thereon. The nano-microsphere successfully achieves immobilization and has the characteristics of repeated use. However, the relative enzyme activity decreases to 70% after repeated use for 7 times. Therefore, it is urgent to find a method for efficiently preparing immobilized D-psicose-3-epimerase with stable enzyme activity and multiple reuse. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for efficiently immobilizing D-psicose-3-epimerase and its application in the preparation of D-psicose, aiming to solve the technical problem of the lack of a method for efficiently preparing immobilized D-psicose-3-epimerase with stable enzyme activity and capable of repeated use.

[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 for 6 hours. After the reaction is completed, wash it with pure water several times and then disperse it in an appropriate amount of pure water. While stirring, slowly add an appropriate amount of 50% glutaraldehyde and stir for 3 hours to obtain aldehyde-modified magnetic nanoparticles. After the reaction is completed, the magnet is recovered and washed with pure water for 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. After the reaction, the magnetic nanoparticles were 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, the magnetic nanoparticles prepared in (1) are added in a ratio of (30:1:10) to (60:1:10) (w / v / v) with 3-aminopropyltriethoxysilane and ethanol, and in a ratio of (10:1:1) to (20:1:1) (w / v / v) with pure water and 50% glutaraldehyde.

[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 highly efficient immobilized enzyme, which comprises mixing and incubating an enzyme with a histidine tag with the immobilized carrier described in the second technical solution.

[0020] In one embodiment, the enzymes include D-psicose-3-epimerase, L-rhamnokinase, and polyphosphate kinase.

[0021] 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.

[0022] In one embodiment, the enzyme with a histidine tag is mixed with the immobilized carrier in a mass ratio of (1:20) to (1:50).

[0023] In one embodiment, the incubation time is 15 to 120 minutes.

[0024] The fourth technical solution provided by the present invention is an immobilized D-psicose-3-epimerase, which is prepared by immobilizing D-psicose-3-epimerase using the method described in the third technical solution.

[0025] The fifth technical solution provided by the present invention is a method for preparing D-psicose, which uses D-fructose as a substrate, utilizes the immobilized D-psicose-3-epimerase to isomerize D-fructose as a catalyst to form a catalytic system, and catalytically obtains D-psicose.

[0026] In one embodiment, in the catalytic system, the added mass ratio of D-fructose and immobilized D-psicose-3-epimerase is 4:1.

[0027] In one embodiment, in the catalytic system, the concentration of D-fructose is 5-40 mg / mL, and the concentration of immobilized D-psicose-3-epimerase is 4 mg / mL.

[0028] In one embodiment, the parameters of the catalytic reaction are as follows: pH 7-9.5, 25-75°C and 5 mM Mn 2+ The reaction was continued for 15 min.

[0029] 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 immobilized D-psicose-3-epimerase described in the fourth technical solution, or the method for preparing D-psicose described in the fifth technical solution in the fields of medicine, food and daily necessities.

[0030] 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 immobilized D-psicose-3-epimerase described in the fourth technical solution, or the method for preparing D-psicose described in the fifth technical solution in preparing a product containing D-psicose.

[0031] The technical effects of the present invention are as follows:

[0032] 1. The method for immobilizing D-psicose-3-epimerase provided by the present invention uses magnetic nanoparticles coated with a zeolite imidazole skeleton as a carrier, connects the enzyme on the surface of the magnetic nanoparticles through affinity binding, and the enzyme activity retention rate of the immobilized D-psicose-3-epimerase can reach up to 100.7%, and the stability of the enzyme can be effectively improved.

[0033] 2. The immobilized enzyme prepared by the present invention can realize repeated use of D-psicose-3-epimerase. Under the optimal environmental conditions, after repeated operation for 20 times, the enzyme activity still maintains more than 67% of the original activity.

[0034] 3. The immobilized D-psicose-3-epimerase prepared by the present invention has higher thermal stability than the free enzyme; after being in a water bath at 55°C for 6 hours, the relative enzyme activity of the free enzyme is 26.8%, while the relative enzyme activity of the immobilized enzyme is more than 60%; after being in a water bath at 65°C for 6 hours, the relative enzyme activity of the free enzyme is completely lost, while the immobilized enzyme still retains more than 34% of the relative enzyme activity.

[0035] 4. The immobilized D-psicose-3-epimerase prepared by the present invention can be used for the preparation of D-psicose, has a fast reaction rate, and can be recycled multiple times, thereby reducing the preparation cost. At the same time, the preparation environment is mild and environmentally friendly, and the conversion rate of D-psicose can reach about 30%.

[0036] 5. The immobilized L-rhamnose kinase prepared by the present invention has good heat resistance and pH stability. At 50°C, the free L-rhamnose kinase has only 37% relative enzyme activity, while the immobilized enzyme retains more than 85% relative enzyme activity.

[0037] 6. The immobilized polyphosphate kinase prepared by the present invention has higher thermal stability than the free enzyme; after being placed in a water bath at 35°C for 2 hours, the relative enzyme activity of the immobilized enzyme reaches more than 105%, while the relative enzyme activity of the free enzyme is only 35%.

[0038] In summary, the present invention provides a novel immobilized carrier, which is a magnetic nanoparticle coated with a zeolite imidazole skeleton and has the function of NTA functionalization to achieve affinity binding. The present invention uses the principle of affinity binding of histidine tag and nitrilotriacetic acid for the first time to immobilize D-psicose-3-epimerase, which can effectively improve the stability of the enzyme and achieve multiple reuses. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a graph showing the effect of different DPE concentrations on the immobilization rate and enzyme activity retention rate.

[0040] Figure 2 VSM diagram of carrier and immobilized DPE.

[0041] Figure 3 FT-IR spectra of carrier and immobilized DPE.

[0042] Figure 4 The figure is a process flow chart for efficient enzyme immobilization.

[0043] Figure 5 This is the effect of time on the DPE solid loading rate.

[0044] Figure 6 This is the effect of pH value on DPE activity.

[0045] Figure 7 This is a graph showing the effect of temperature on DPE activity.

[0046] Figure 8 The reusability of the prepared immobilized DPE.

[0047] Fig. 9 The thermal stability diagram of immobilized DPE and free enzyme at 45℃, 55℃, and 65℃.

[0048] Fig.10 This is a graph showing the effect of time on the conversion rate of D-psicose.

[0049] Fig.11 This is a graph showing the effect of pH on rhab activity.

[0050] Fig.12 This is a graph showing the effect of temperature on rhab activity.

[0051] Fig.13 This is a graph showing the effect of temperature on ppk activity.

[0052] Fig.14 This is the effect of pH on ppk activity.

[0053] Fig.15 This is the thermal stability diagram of immobilized ppk. DETAILED DESCRIPTION

[0054] 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.

[0055] Test method:

[0056] 1. Enzyme activity detection of D-psicose-3-epimerase: After the reaction, the reaction solution was transferred to a boiling water bath for 5 minutes. After cooling, it was centrifuged at 10,000 rpm for 1 minute. After that, it was filtered through a 0.22 μm filter membrane and the amount of D-psicose generated was detected by high performance liquid chromatography. One enzyme activity unit (U) was defined as 1 μmol of D-psicose generated per mg of enzyme per minute.

[0057] 2. Detection of D-psicose by high performance liquid chromatography: Waters high performance liquid chromatograph, e2414 differential detector, COSMOSIL Sugar-D 4.6 mm×250 mm chromatographic column; the mobile phase was 80% acetonitrile, the flow rate was 1.0 ml / min, and the column temperature was 30°C.

[0058] Materials used in the examples:

[0059] 1. E. coli BL21 (DE3) / pET22b-dpe strain has been disclosed in the following literature: 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.

[0060] 2. pET28a-ppk was published in: Feng Linxue. Research on the synthesis of D-psicose from D-fructose catalyzed by multiple enzymes [D]. Jiangnan University, 2021.

[0061] 3. LB liquid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L;

[0062] LB solid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L;

[0063] The culture medium was prepared with deionized water and sterilized at 121°C for 20 min.

[0064] 4. Nα, Nα-bis(carboxymethyl)-L-lysine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0065] Example 1 Preparation of immobilized carrier

[0066] The specific steps are as follows:

[0067] 1. Preparation of zeolite imidazole framework-8 (ZIF-8) coated magnetic nanoparticles: 300 mg Fe3O4 (200 nm) was dissolved in 200 mL polystyrene sodium sulfonate (PSS) solution (0.3%, w / v), then ultrasonicated and stirred evenly at room temperature, and the nanoparticles were recovered with a magnet after 30 minutes, 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 minutes, and then 4.2 g 2-methylimidazole was slowly added to the solution and stirred for 4 hours. After the end, the magnet was recovered and washed with ethanol 3 to 4 times and then dried at 40°C to 50°C for 12 hours.

[0068] 2. Aldehyde modification: ultrasonically disperse the magnetic nanoparticles prepared in the first step in 40 mL of ethanol, then add 6 mL of 3-aminopropyltriethoxysilane (APTES) and stir for 6 hours; after the reaction is completed, wash with pure water several times and disperse in 30 mL of pure water, slowly add 30 mL of 50% glutaraldehyde while stirring, and stir for 3 hours to obtain aldehyde-modified magnetic nanoparticles. After the reaction is completed, the magnet is recovered and washed with pure water for 3 to 4 times.

[0069] 3. Functionalization with nitrilotriacetic acid (NTA): Ultrasonic dispersion of the washed magnetic nanoparticles in the second step in 60 mL pure water, add 12 mL 50 mM, pH 8 (50 mM PBS) Nα, Nα-bis (carboxymethyl) -L-lysine hydrate solution, stir for 24 hours, and wash with ultrapure water 3 to 4 times after the reaction. Resuspend the washed magnetic nanoparticles in 60 mL NiCl2 (50 mM) 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 magnetic nanoparticles functionalized with nitrilotriacetic acid.

[0070] 4. Immobilization rate and enzyme activity retention rate: Different concentrations of D-psicose-3-epimerase (0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL) were mixed with 1 mL of 50 mg / mL carrier and immobilized. Figure 1It can be found that the immobilization rate did not change significantly under the conditions of 0.4-1 mg / mL, and the enzyme activity retention rate reached the highest under the conditions of 1 mg / mL.

[0071] 5. Characterization of immobilized carrier and immobilized enzyme: Figure 2 VSM shows that the immobilized material exhibits paramagnetism. The saturation magnetic intensity of Fe3O4 to DPE-ZIF-8@Fe3O4 decreases from 77.36emu / g to 74.26emu / g. The immobilized enzyme reduces the overall saturation magnetic intensity. Figure 3 It can be seen from the Fourier infrared spectrum that NTA-functionalized magnetic nanoparticles were successfully synthesized and DPE was successfully immobilized on the carrier.

[0072] Example 2 Preparation of immobilized D-psicose-3-epimerase

[0073] like Figure 4 As shown, the specific steps are as follows:

[0074] (1) Preparation of dpe: E. coli BL21 (DE3) / pET22b-dpe was cultured in LB medium at 37 ° C constant temperature shaker for about 2 h, 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 1 mL Ni 2+ The crude enzyme solution was purified by affinity chromatography on the column to obtain dpe with an enzyme activity of 30-40 U / mg;

[0075] (2) Immobilized D-psicose-3-epimerase:

[0076] The purified DPE was ultrafiltered to remove imidazole, and the ultrafiltration cut-off molecular weight was 10KDa. Then the enzyme concentration was adjusted to 1 mg / mL, and the concentration of the immobilized carrier prepared in Example 1 was adjusted to 50 mg / mL. 1 ml of enzyme solution was mixed with 1 ml of carrier and fixed at 16°C for 15 to 120 minutes. Then the supernatant was removed, washed with deionized water 3 times, and finally resuspended with 2 ml of deionized water. Figure 5 As shown in the figure, a solid loading rate of 95% can be achieved in 15 min.

[0077] Example 3 Enzymatic properties of immobilized D-psicose-3-epimerase

[0078] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8.5), 50 μL of immobilized D-psicose-3-epimerase and free enzyme prepared in Example 2 were added, and the enzyme activities were measured after reacting for 15 min and 5 min, respectively.

[0079] 1. Effect of pH on enzyme activity:

[0080] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ ), 50 μL of immobilized D-psicose-3-epimerase and free enzyme prepared in Example 1 were added, and the mixture was reacted at pH 7-9.5 for 15 min and 5 min, respectively, and the enzyme activities were measured.

[0081] from Figure 6 It can be seen that the optimum pH for both the immobilized enzyme and the free enzyme is 8.5.

[0082] 2. Effect of temperature on enzyme activity:

[0083] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ ), 50 μL of immobilized D-psicose-3-epimerase and free enzyme prepared in Example 1 were added, and the mixture was reacted at pH 8.5 and 25-75° C. for 15 min and 5 min, respectively, and the enzyme activity was measured.

[0084] from Figure 7 It can be seen that the optimal reaction temperatures of free enzyme and immobilized enzyme are 65℃ and 55℃, respectively.

[0085] 3. Reusability of immobilized enzyme:

[0086] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ ), 50 μL of immobilized D-psicose-3-epimerase prepared in Example 1 was added, and the mixture was reacted at pH 8.5 and 55°C for 15 min. The immobilized enzyme was collected by magnet and then added to 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8.5) and cycled 20 times.

[0087] from Figure 8 It can be seen that, with the enzyme activity at the first use as 100%, after 20 cycles of repeated operation of the immobilized enzyme, the immobilized enzyme still retained 67% of the original activity. This shows that the immobilized D-psicose-3-epimerase can be effectively reused multiple times.

[0088] Example 4 Thermal stability of immobilized D-psicose-3-epimerase

[0089] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+, pH 8.5), added 50 μL of immobilized D-psicose-3-epimerase and free enzyme prepared in Example 2, reacted for 15 min and 5 min, respectively, and measured the enzyme activities.

[0090] Thermal stability of immobilized and free enzymes:

[0091] from Fig. 9 It can be seen that with the increase of 55℃ water bath time, the enzyme activities of both free enzyme and immobilized enzyme gradually decreased, but the enzyme activity loss rate of immobilized D-psicose-3-isomerase was significantly lower than that of free enzyme. After 6h of water bath, the relative enzyme activities of free enzyme and immobilized enzyme were 26.8% and 60%, respectively. After 6h of 65℃, the relative enzyme activity of immobilized enzyme decreased to 33.8%, while the free enzyme completely lost its enzyme activity.

[0092] Example 5 Preparation of D-psicose

[0093] Take 200 μL of substrate solution (20 mg / mL fructose, 5 mM Mn 2+ , pH 8.5), 50 μL of immobilized D-psicose-3-epimerase prepared in Example 2 was added, and the reaction was carried out for 3, 5, 10, 15, 20, 30, and 40 min, respectively, and the D-psicose conversion rate was measured. Fig.10 It can be seen that with the increase of reaction time, the conversion rate of D-psicose gradually decreased, and finally reached equilibrium at 40 min, with a conversion rate of about 30%.

[0094] Example 6 Preparation of immobilized L-rhamnokinase

[0095] (1) Preparation of rhab enzyme solution: Using 6×His-tag as the C-terminal fusion peptide of the protein, Rhab (genebank: CAD6021149.1) was inserted between the BmHⅠ and XhoⅠ restriction sites of plasmid pET28a, and the recombinant plasmid pET28a-rhab was constructed. The plasmid pET28a-rhab was transformed into Escherichia coli E.coli BL21(DE3) to obtain the recombinant bacteria E.coli BL21(DE3) / pET28a-rhab. The recombinant bacteria E.coli BL21(DE3) / pET28a-rhab were inoculated into LB culture medium, cultured in a constant temperature shaker at 37°C for about 2 hours, and then IPTG with a final concentration of 1mM was added to induce expression, and the bacteria were collected by centrifugation. The bacteria were resuspended in buffer, disrupted with an ultrasonic disruptor, and the supernatant was collected by centrifugation to obtain the crude enzyme solution. Then His Trap HP 1mL Ni 2+ The crude enzyme solution was purified by affinity chromatography using a column to obtain rhab;

[0096] (2) Immobilized L-rhamnokinase:

[0097] The purified rhab was 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.

[0098] Example 7 Enzymatic properties of immobilized L-rhamnose glycokinase

[0099] Take 200 μL of substrate solution (20 mg / mL allulose, 5 mM Mn 2+ , pH 8, 100 mM ATP), 50 μL of free enzyme and immobilized enzyme prepared in Example 6 were added, reacted for 5 min and 15 min respectively, and the enzyme activities were measured.

[0100] 1. Effect of pH on enzyme activity:

[0101] Take 200 μL of substrate solution (20 mg / mL allulose, 5 mM Mn 2+ , 100 mM ATP), add 50 μL of immobilized L-rhamnose kinase and free enzyme prepared in Example 4, react for 15 min and 5 min respectively at pH 6.5-9, and measure the enzyme activity. Fig.11 It can be seen that the optimum pH for both the immobilized enzyme and the free enzyme is 8.

[0102] 2. Effect of temperature on enzyme activity:

[0103] Take 200 μL of substrate solution (20 mg / mL allulose, 5 mM Mn 2+ , 100 mM ATP), add 50 μL of immobilized L-rhamnose kinase and free enzyme prepared in Example 4, react at pH 8, 30-50°C for 15 min and 5 min respectively, and measure the enzyme activity. Fig.12 It can be seen that the optimal reaction temperature of free enzyme and immobilized enzyme is 40℃.

[0104] Example 8 Preparation of immobilized polyphosphate kinase

[0105] (1) Preparation of ppk enzyme solution: The plasmid pET28a-ppk was transformed into E. coli BL21 (DE3) to obtain the recombinant bacteria E. coli BL21 (DE3) / pET28a-ppk. The recombinant bacteria E. coli BL21 (DE3) / pET28a-ppk 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 bacteria were collected by centrifugation. The bacteria were resuspended in buffer, crushed with an ultrasonic disruptor, and the supernatant was collected by centrifugation to obtain the crude enzyme solution. Then His Trap HP1mL Ni 2+ The crude enzyme solution was purified by affinity chromatography using a column to obtain ppk;

[0106] (2) Immobilized polyphosphate kinase:

[0107] The purified ppk was 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.

[0108] Example 9 Enzymatic properties of immobilized polyphosphate kinase

[0109] Take 200 μL substrate solution (20 mM ATP, 5 mM Mn 2+ , pH 8, 10 mM Poly6), 50 μL of free enzyme and immobilized enzyme prepared in Example 5 were added, reacted for 5 min and 15 min respectively, and the enzyme activities were measured.

[0110] 1. Effect of pH on enzyme activity:

[0111] Take 200 μL substrate solution (20 mM ATP, 5 mM Mn 2+ , pH 8, 10 mM Poly6), 50 μL of immobilized polyphosphate kinase and free enzyme prepared in Example 5 were added, and the reaction was carried out at pH 7-9 for 15 min and 5 min, respectively, and the enzyme activity was measured. Fig.13 It can be seen that the optimum pH for both the immobilized enzyme and the free enzyme is 7.5.

[0112] 2. Effect of temperature on enzyme activity:

[0113] Take 200 μL substrate solution (20 mM ATP, 5 mM Mn 2+, pH 8, 10 mM Poly6), add 50 μL of immobilized L-rhamnose kinase and free enzyme prepared in Example 5, react at pH 8, 30-50°C for 15 min and 5 min respectively, and measure the enzyme activity. Fig.14 It can be seen that the optimal reaction temperatures of free enzyme and immobilized enzyme are 30℃ and 35℃, respectively.

[0114] 3. Heat resistance of immobilized polyphosphate kinase

[0115] Take 200 μL substrate solution (20 mM ATP, 5 mM Mn 2+ , pH 8, 10 mM Poly6), add 50 μL of immobilized L-rhamnose kinase and free enzyme prepared in Example 5, incubate at pH 8, 35-45°C for 0-120 min, react for 15 min and 5 min, and measure the enzyme activity. Fig.15 It can be seen that after the free enzyme was incubated at 35°C for 120 min, the relative enzyme activity remained about 35%, while the immobilized enzyme was able to retain 105% of the relative enzyme activity after being incubated at 35°C for 120 min.

[0116] 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 an immobilized carrier, characterized in that: The method comprises the following steps: covering the surface of Fe3O4 nanoparticles with a shell of a zeolite imidazole skeleton, 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, namely, immobilized carriers.

2. The method according to claim 1, characterized in that The steps include: (1) Preparation of zeolite imidazole framework-8 coated magnetic nanoparticles: Fe3O4 was dissolved in a sodium polystyrene sulfonate solution, and then ultrasonicated and stirred at room temperature, and the nanoparticles were recovered by a magnet, washed with methanol, and then the magnetic nanoparticles were dispersed in methanol; an appropriate amount of soluble zinc salt was added to the solution and stirred, and then 2-methylimidazole was slowly added to the solution and stirred, and after completion, 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) Functionalization with nitrilotriacetic acid: The magnetic nanoparticles washed in (2) were ultrasonically dispersed in pure water, and a 50 mM, pH 8 Nα, Nα-bis(carboxymethyl)-L-lysine hydrate solution was added and stirred. After the reaction, the magnetic nanoparticles were washed with ultrapure water, and then resuspended in a 50 mM NiCl2 aqueous solution and stirred. After the reaction, the magnetic nanoparticles were washed with pure water and dried at low temperature to obtain nitrilotriacetic acid-functionalized magnetic nanoparticles.

3. The method according to claim 2, characterized in that In step (1), the particle size of Fe3O4 is 200nm, the concentration of the sodium polystyrene sulfonate solution is 0.3-0.5% w / v; the addition ratio of Fe3O4 to 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); In step (2), the magnetic nanoparticles prepared in (1) are added to 3-aminopropyltriethoxysilane and ethanol in a ratio of (30:1:10) to (60:1:10) (w / v / v), and to pure water and 50% glutaraldehyde in a ratio of (10:1:1) to (20:1:1) (w / v / v); In step (3), the ratio of the magnetic nanoparticles prepared in (2) to pure water and Nα,Nα-bis(carboxymethyl)-L-lysine hydrate solution is (20:5:1)-(40:5:1) (w / v / v), and the ratio of the magnetic nanoparticles prepared in (2) to the NiCl2 aqueous solution is (4:1)-(6:1) (w / v); The temperature of the low-temperature drying is 40° C. to 50° C., and the time of the low-temperature drying is 12 hours.

4. An immobilized carrier prepared by the method described in any one of claims 1 to 3.

5. A method for preparing a highly efficient immobilized enzyme, characterized in that: The method comprises mixing the enzyme with the histidine tag with the immobilized carrier according to claim 4 and incubating the mixture.

6. The method according to claim 5, characterized in that: The enzymes include D-psicose-3-epimerase, L-rhamnokinase, and polyphosphate kinase.

7. The method according to claim 5, characterized in that: Mixing the enzyme with the histidine tag with the immobilized carrier at a mass ratio of (1:20) to (1:50); The incubation time is 15 to 120 minutes.

8. An immobilized D-psicose-3-epimerase, characterized in that: The D-psicose-3-epimerase is immobilized by the method according to claim 5 or 7.

9. A method for preparing D-psicose, characterized in that: The method comprises taking D-fructose as a substrate and utilizing the immobilized D-psicose-3-epimerase as a catalyst to isomerize D-fructose to form a catalytic system, thereby catalytically obtaining D-psicose.

10. Use of the method according to any one of claims 1 to 3, or the immobilized carrier according to claim 4, or the method according to any one of claims 5 to 7, or the immobilized D-psicose-3-epimerase according to claim 8, or the method according to claim 9 in preparing a product containing D-psicose.

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