Method for preparing psicose by catalyzing fructose conversion through immobilized isomerase

Abstract

The invention discloses a method for preparing psicose by catalyzing fructose conversion through immobilized isomerase, and belongs to the technical field of sweeteners, the method comprises the following steps: glycosylation of DAEase enzyme, preparation of a lectin carrier, immobilization of glycosylated DAEase enzyme, and catalytic preparation of psicose; the method for immobilizing the glycosylated DAEase enzyme comprises the following steps: dispersing the glycosylated DAEase enzyme and a lectin carrier in a sodium phosphate buffer solution, stirring at room temperature for 1-1.2 hours, centrifuging to remove supernate, adding into a glutaraldehyde solution, cross-linking at room temperature for 1-1.5 hours, and freeze-drying to obtain the immobilized isomerase, according to the method disclosed by the invention, the activity of the immobilized enzyme, the conversion rate of D-fructose and the catalytic rate of DAEase are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of sweeteners, and particularly relates to a method for preparing allulose by immobilizing isomerase to catalyze fructose conversion. BACKGROUND

[0002] D-allulose has a molecular weight of 180.16 and a melting point of 96℃, is easily soluble in water, has a sweetness of about 70% of sucrose, and is different from D-fructose in the C-3 position. In addition to having low energy, improving intestinal flora, and anti-caries physiological functions, D-allulose also plays an important physiological role in inhibiting blood glucose rise and body fat accumulation, scavenging free radicals, and neuroprotection. In addition, D-allulose can be used to make environmentally friendly, waterproof, and transparent liquid crystal display films and optical devices, and as a cosmetic raw material, has skin moisturizing and anti-acid effects.

[0003] D-allulose naturally exists in nature in extremely small amounts and is mainly derived from a small number of plants and a small number of bacteria. There are two main methods for synthesizing D-allulose: chemical synthesis and biological conversion. The chemical synthesis method has the problems of complex purification steps, more by-products, serious environmental pollution, and low stereoselectivity; compared with the chemical synthesis method, the biological conversion method is easy to operate and has no other by-products.

[0004] The biological conversion method mainly includes two methods: (1) using dihydroxyacetone phosphate (DHAP)-dependent aldolase to synthesize D-allulose, DHAP can react with D / L-glyceraldehyde to form a phosphosugar under the action of aldolase, and then be dephosphorylated into various rare sugars such as D / L-allulose, D / L-tagatose, etc. by the action of phosphatase; (2) using Izumoring strategy to synthesize D-allulose. D-allulose is usually synthesized by using D-fructose as a substrate and D-allulose 3-epimerase (DAEase enzyme) to catalyze the conversion of D-fructose to D-allulose.

[0005] In recent years, researchers have mined, characterized, and modified various DAEase enzymes from different sources, but the thermal stability of these DAEase enzymes still cannot meet the continuous high-temperature requirements of industrial production. Compared with free enzymes, immobilized DAEase has good recovery performance and can be repeatedly used in the application process, and has certain advantages in large-scale industrial production.

[0006] The enzyme immobilization methods include adsorption method, embedding method, covalent binding and the like, the adsorption method has simple preparation process, low cost and less mass transfer limitation, but this technology usually has low biological catalyst loading, and the adsorbed material is easy to leach out; the embedding method provides a protective shell for the biological catalyst, and the macromolecule can be significantly limited in mass transfer by the shell; the covalent binding method forms a covalent bond through the amino, carboxyl and the like on the surface of the enzyme molecule and the active groups (such as epoxy group, aldehyde group) of the carrier to realize firm fixation, but this process has two problems: one is that the reaction site of covalent binding is random - the active center of DAEase enzyme is composed of key residues such as His9, Glu146 and Glu152, and lysine and other amino acid residues containing amino groups are usually distributed around the active center, if covalent binding occurs on the amino group near the active center, the active site will be directly shielded by the modification group; the other is that the formation of covalent bond is easy to cause the conformation distortion of enzyme molecules - even if the binding site is far away from the active center, the spatial steric hindrance of the covalent bond can also cause the change of secondary or tertiary structure of the enzyme molecules to make the spatial conformation of the active center close, so that the substrate cannot enter the catalytic pocket.

[0007] In summary, although the immobilization technology is the key to solve the industrial application of DAEase enzyme, the "randomness" of the existing immobilization method leads to the shielding of the enzyme active center, therefore, developing a technical scheme capable of realizing the specific positioning and fixation of DAEase enzyme and ensuring the sufficient exposure of the active center has important significance for promoting the efficient industrial production of D-allulose. SUMMARY

[0008] In view of the defects in the prior art, the application provides a method for preparing allulose by catalyzing the conversion of fructose through immobilized isomerase, which improves the activity of the immobilized enzyme, the conversion rate of D-fructose and the catalytic rate of DAEase enzyme.

[0009] To solve the above technical problems, the technical scheme adopted by the application is as follows: A method for preparing allulose by catalyzing the conversion of fructose through immobilized isomerase comprises the following steps: glycosylation of DAEase enzyme, preparation of a lectin carrier, fixation of glycosylated DAEase enzyme and catalytic preparation of allulose. The glycosylation of DAEase enzyme is to introduce a sugar chain on the DAEase enzyme to obtain glycosylated DAEase enzyme. The glycosylation method of DAEase enzyme is to dilute DAEase enzyme and aldehyde-mannose into DAEase enzyme solution and aldehyde-mannose solution respectively with sodium phosphate buffer solution, mix the two solutions uniformly, add 2-methylpyridine borane solution after 6-8 min, react in the dark at 3-5 DEG C for 4-5 h, and obtain glycosylated DAEase enzyme after dialysis. The DAEase enzyme is derived from thermophilic bacillus. The molecular weight of the DAEase enzyme is 50,000-100,000 Da; The concentration of the DAEase enzyme solution is 2-2.5 g / L; The concentration of the aldehyde-modified mannose solution is 2.5-3.5 g / L; The aldehyde-modified mannose is aldehyde-D-mannose; The 2-methylpyridineborane solution is obtained by dissolving 2-methylpyridineborane in sodium phosphate buffer solution, with a concentration of 4-5 g / L; The concentration of the sodium phosphate buffer solution is 0.08-0.12 mol / L, and the pH is 6.8-7.2. The mass ratio of the DAEase enzyme solution, the aldehyde-modified mannose solution, and the 2-methylpyridineborane solution is 10:1.2-1.4:4-6.

[0010] The method for preparing the lectin carrier is as follows: aminated porous microspheres are dispersed in a lectin solution, incubated by rotation at 3-5°C for 22-24 hours, washed with phosphate buffer, and then freeze-dried to obtain the lectin carrier. The lectin is concanavalin A; The concanavalin A solution is obtained by dissolving concanavalin A in sodium phosphate buffer solution, and contains 18-22 mg / mL concanavalin A. The aminated microspheres are amino-porous silica microspheres with a particle size of 200-500 μm, a pore size of 50-100 nm, and a porosity of 60-70%. The amino loading of the aminated microspheres is 2.8-3.2 mmol / g; The mass ratio of the aminated microspheres to the canavanine A solution is 1:18-22. The sodium phosphate buffer solution has a concentration of 0.08-0.12 mol / L and a pH of 6.8-7.2.

[0011] The method for immobilizing glycosylated DAEase enzyme is as follows: glycosylated DAEase enzyme and lectin carrier are dispersed in sodium phosphate buffer, stirred at room temperature for 1-1.2 h, centrifuged to remove supernatant, added to glutaraldehyde solution, crosslinked at room temperature for 1-1.5 h, and freeze-dried to obtain immobilized isomerase. The concentration of the sodium phosphate buffer solution is 0.08-0.12 mol / L, and the pH is 6.8-7.2. The mass ratio of the glycosylated DAEase enzyme, lectin carrier, sodium phosphate buffer, and glutaraldehyde solution is 0.15:4-5:10-15:8-12. The concentration of the glutaraldehyde solution is 0.6-0.8 g / L.

[0012] The method for catalytically preparing allulose involves adding an immobilized isomerase to a fructose solution containing 1-2 mmol / L manganese chloride, and stirring the reaction at 68-72°C and pH 7.4-7.6 until equilibrium is reached. The concentration of the fructose solution is 450-550 g / L; The mass ratio of the immobilized isomerase to the fructose solution is 1:30-40; The stirring speed is 300-400 rpm; The equilibrium state is when the concentrations of fructose and allulose in the mixture no longer change; The method for determining the equilibrium state is as follows: after the reaction has been going on for 1 hour, the concentrations of fructose and allulose in the solution are measured every 15 minutes until the concentration fluctuation is less than 5%, which is considered as reaching the reaction equilibrium.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the glycosylation step of the present invention, the pKa value of the N-terminal α-amino group of the DAEase enzyme is about 7.6, which is lower than that of the ε-amino group of the lysine (Lys) side chain (pKa ~ 10.5). By controlling the reaction pH under mild and neutral conditions, the N-terminal amino group has a low degree of protonation and exhibits high nucleophilic activity. The aldehyde-modified mannose preferentially reacts with the N-terminal amino group of the most reactive site to generate a Schiff base product. By controlling the reaction time, other sites do not have time to react. Then, the generated Schiff base (C=N-) is reduced to a stable secondary amine (CN-) by a reducing agent that is added quickly, thus completing the glycosylation and introducing a sugar chain onto the DAEase enzyme.

[0014] Lectin can specifically recognize sugar chains such as mannose, glucose, and N-acetylglucosamine. After the porous microspheres are modified with lectin, the sugar chains of glycosylated DAEase enzyme are bound by lectin, completing specific localization and exposing the active site of DAEase enzyme. The cross-linking of glutaraldehyde forms strong, covalent chemical bonds between lectin, enzyme molecules, and carrier surfaces, preventing the immobilized enzyme from falling off under high-temperature operating conditions. The sugar-binding domain of lectins can specifically recognize glycosyl groups on the enzyme surface and fix the enzyme through non-covalent or weak covalent interactions (such as hydrogen bonds and van der Waals forces), avoiding strong covalent bonds from directly acting on key regions of the enzyme molecule and reducing the risk of conformational distortion.

[0015] This invention specifically exposes the active site of the immobilized DAEase enzyme, thereby increasing the activity of the immobilized DAEase enzyme, improving the conversion rate of D-fructose, and increasing the catalytic rate of the DAEase enzyme.

[0016] (2) The method of preparing allulose by fructose conversion catalyzed by immobilized isomerase of the present invention has a reaction equilibrium time of 270-285 min and a conversion rate of 26.4-27.2%. Detailed Implementation

[0017] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0018] Example 1 A method for preparing allulose by catalyzing the conversion of fructose using an immobilized isomerase includes the following steps: (1) Glycosylation of DAEase DAEase enzyme and aldehyde-D-mannose were diluted with sodium phosphate buffer to prepare DAEase enzyme solution of 2 g / L and aldehyde-D-mannose solution of 2.5 g / L, respectively. The two solutions were mixed evenly, and after 6 min, 4 g / L 2-methylpyridineborane solution was added. The mixture was reacted at 3°C ​​for 5 h in the dark. After dialysis, glycosylated DAEase enzyme was obtained. The DAEase enzyme is derived from Bacillus thermophilus and has a molecular weight of 50,000 Da. The 2-methylpyridineborane solution is obtained by dissolving 2-methylpyridineborane in sodium phosphate buffer solution; The sodium phosphate buffer solution has a concentration of 0.08 mol / L and a pH of 6.8. The mass ratio of the DAEase enzyme solution, the aldehyde-modified mannose solution, and the 2-methylpyridineborane solution is 10:1.2:4.

[0019] (2) Preparation of lectin carriers Aminated porous microspheres were dispersed in concanavalin A solution, incubated at 3°C ​​for 24 h by rotation, washed with phosphate buffer, and then freeze-dried to obtain the lectin carrier. The concanavalin A solution was obtained by dissolving concanavalin A in sodium phosphate buffer solution, with a concentration of 18 mg / mL. The aminated microspheres are amino porous silica microspheres with a particle size of 200 μm, a pore size of 50 nm, and a porosity of 60%. The amino loading of the aminated microspheres is 2.8 mmol / g; The mass ratio of the aminated microspheres to the canavanine A solution is 1:18.

[0020] (3) Immobilized glycosylation DAEase enzyme 0.3g of glycosylated DAEase enzyme and 8g of lectin carrier were dispersed in 20g of sodium phosphate buffer, stirred at room temperature for 1h, centrifuged to remove the supernatant, added to 16g of glutaraldehyde solution, crosslinked at room temperature for 1h, and freeze-dried to obtain immobilized isomerase. The sodium phosphate buffer solution has a concentration of 0.08 mol / L and a pH of 6.8. The concentration of the glutaraldehyde solution is 0.6 g / L.

[0021] (4) Catalytic preparation of allulose 1g of immobilized isomerase was added to 30g of a 550g / L fructose solution containing 1mmol / L manganese chloride. The mixture was stirred at 68℃, pH 7.4, and 300rpm until equilibrium was reached. The equilibrium state is when the concentrations of fructose and allulose in the mixture no longer change; The method for determining the equilibrium state is as follows: after the reaction has been going on for 1 hour, the concentrations of fructose and allulose in the solution are measured every 15 minutes until the concentration fluctuation is less than 5%, which is considered as reaching the reaction equilibrium.

[0022] In Example 1, the time to reach equilibrium was 285 minutes, and the fructose conversion rate was 26.7%.

[0023] Example 2 A method for preparing allulose by catalyzing the conversion of fructose using an immobilized isomerase includes the following steps: (1) Glycosylation of DAEase DAEase enzyme and aldehyde-D-mannose were diluted with sodium phosphate buffer to prepare DAEase enzyme solution of 2.2 g / L and aldehyde-D-mannose solution of 3 g / L, respectively. The two solutions were mixed evenly, and after 7 min, 4.5 g / L 2-methylpyridineborane solution was added. The mixture was reacted in the dark at 4 °C for 4.5 h. After dialysis, glycosylated DAEase enzyme was obtained. The DAEase enzyme is derived from Bacillus thermophilus and has a molecular weight of 80,000 Da. The 2-methylpyridineborane solution is obtained by dissolving 2-methylpyridineborane in sodium phosphate buffer solution; The sodium phosphate buffer solution has a concentration of 0.1 mol / L and a pH of 7. The mass ratio of the DAEase enzyme solution, the aldehyde-modified mannose solution, and the 2-methylpyridineborane solution is 10:1.3:5.

[0024] (2) Preparation of lectin carriers Aminated porous microspheres were dispersed in concanavalin A solution, incubated at 4°C for 23 h by rotation, washed with phosphate buffer, and then freeze-dried to obtain the lectin carrier. The concanavalin A solution was obtained by dissolving concanavalin A in sodium phosphate buffer solution, with a concentration of 20 mg / mL. The aminated microspheres are amino-porous silica microspheres with a particle size of 300 μm, a pore size of 80 nm, and a porosity of 65%. The amino loading of the aminated microspheres is 3 mmol / g; The mass ratio of the aminated microspheres to the canavanine A solution is 1:20.

[0025] (3) Immobilized glycosylation DAEase enzyme 0.3 g of glycosylated DAEase enzyme and 9 g of lectin carrier were dispersed in 25 g of sodium phosphate buffer and stirred at room temperature for 1.1 h. After centrifugation to remove the supernatant, 20 g of glutaraldehyde solution was added and crosslinked at room temperature for 1.2 h. The immobilized isomerase was obtained by freeze drying. The sodium phosphate buffer solution has a concentration of 0.1 mol / L and a pH of 7. The concentration of the glutaraldehyde solution is 0.7 g / L.

[0026] (4) Catalytic preparation of allulose 1g of immobilized isomerase was added to 35g of a 500g / L fructose solution containing 1.5mmol / L manganese chloride. The mixture was stirred at 70℃, pH 7.5, and 350rpm until equilibrium was reached. The equilibrium state is when the concentrations of fructose and allulose in the mixture no longer change; The method for determining the equilibrium state is as follows: after the reaction has been going on for 1 hour, the concentrations of fructose and allulose in the solution are measured every 15 minutes until the concentration fluctuation is less than 5%, which is considered as reaching the reaction equilibrium.

[0027] In Example 2, the time to reach equilibrium was 270 minutes, and the fructose conversion rate was 27.2%.

[0028] Example 3 A method for preparing allulose by catalyzing the conversion of fructose using an immobilized isomerase includes the following steps: (1) Glycosylation of DAEase DAEase enzyme and aldehyde-D-mannose were diluted with sodium phosphate buffer to prepare DAEase enzyme solution of 2.5 g / L and aldehyde-D-mannose solution of 3.5 g / L, respectively. The two solutions were mixed evenly, and after 8 min, 5 g / L 2-methylpyridineborane solution was added. The mixture was reacted in the dark at 5 °C for 4 h. After dialysis, glycosylated DAEase enzyme was obtained. The DAEase enzyme is derived from Bacillus thermophilus and has a molecular weight of 100,000 Da. The 2-methylpyridineborane solution is obtained by dissolving 2-methylpyridineborane in sodium phosphate buffer solution; The sodium phosphate buffer solution has a concentration of 0.12 mol / L and a pH of 7.2. The mass ratio of the DAEase enzyme solution, the aldehyde-modified mannose solution, and the 2-methylpyridineborane solution is 10:1.4:6.

[0029] (2) Preparation of lectin carriers Aminated porous microspheres were dispersed in concanavalin A solution, incubated at 5°C for 22 h by rotation, washed with phosphate buffer, and then freeze-dried to obtain the lectin carrier. The concanavalin A solution was obtained by dissolving concanavalin A in sodium phosphate buffer solution, with a concentration of 22 mg / mL. The aminated microspheres are amino-porous silica microspheres with a particle size of 500 μm, a pore size of 100 nm, and a porosity of 70%. The amino loading of the aminated microspheres is 3.2 mmol / g; The mass ratio of the aminated microspheres to the canavanine A solution is 1:22.

[0030] (3) Immobilized glycosylation DAEase enzyme 0.3 g of glycosylated DAEase enzyme and 10 g of lectin carrier were dispersed in 30 g of sodium phosphate buffer and stirred at room temperature for 1.2 h. After centrifugation to remove the supernatant, 24 g of glutaraldehyde solution was added and crosslinked at room temperature for 1.5 h. The immobilized isomerase was obtained by freeze drying. The sodium phosphate buffer solution has a concentration of 0.12 mol / L and a pH of 7.2. The concentration of the glutaraldehyde solution is 0.8 g / L.

[0031] (4) Catalytic preparation of allulose 1g of immobilized isomerase was added to 40g of a 450g / L fructose solution containing 2mmol / L manganese chloride. The mixture was stirred at 72℃, pH 7.6, and 400rpm until equilibrium was reached. The equilibrium state is when the concentrations of fructose and allulose in the mixture no longer change; The method for determining the equilibrium state is as follows: after the reaction has been going on for 1 hour, the concentrations of fructose and allulose in the solution are measured every 15 minutes until the concentration fluctuation is less than 5%, which is considered as reaching the reaction equilibrium.

[0032] In Example 3, the time to reach equilibrium was 285 minutes, and the fructose conversion rate was 26.9%.

[0033] Example 4 The preparation of an immobilized isomerase includes the following steps: (1) Glycosylation of DAEase DAEase enzyme and aldehyde-D-mannose were diluted with sodium phosphate buffer to prepare DAEase enzyme solution of 2.2 g / L and aldehyde-D-mannose solution of 3 g / L, respectively. The two solutions were mixed evenly, and after 7 min, 4.5 g / L 2-methylpyridineborane solution was added. The mixture was reacted in the dark at 4 °C for 4.5 h. After dialysis, glycosylated DAEase enzyme was obtained. The DAEase enzyme is derived from Bacillus thermophilus and has a molecular weight of 80,000 Da. The 2-methylpyridineborane solution is obtained by dissolving 2-methylpyridineborane in sodium phosphate buffer solution; The sodium phosphate buffer solution has a concentration of 0.1 mol / L and a pH of 7. The mass ratio of the DAEase enzyme solution, the aldehyde-modified mannose solution, and the 2-methylpyridineborane solution is 10:1.3:5.

[0034] (2) Immobilization of glycosylated DAEase enzyme 0.3 g of glycosylated DAEase enzyme and 9 g of carrier were dispersed in 25 g of sodium phosphate buffer and stirred at room temperature for 1.1 h. After centrifugation to remove the supernatant, 20 g of glutaraldehyde solution was added and crosslinked at room temperature for 1.2 h. The immobilized isomerase was obtained by freeze drying. The carrier is amino-porous silica microspheres with a particle size of 300 μm, a pore size of 80 nm, and a porosity of 65%. The sodium phosphate buffer solution has a concentration of 0.1 mol / L and a pH of 7. The concentration of the glutaraldehyde solution is 0.7 g / L.

[0035] (3) Catalytic preparation of allulose 1g of immobilized isomerase was added to 35g of a 500g / L fructose solution containing 1.5mmol / L manganese chloride. The mixture was stirred at 70℃, pH 7.5, and 350rpm until equilibrium was reached. The equilibrium state is when the concentrations of fructose and allulose in the mixture no longer change; The method for determining the equilibrium state is as follows: after the reaction has been going on for 1 hour, the concentrations of fructose and allulose in the solution are measured every 15 minutes until the concentration fluctuation is less than 5%, which is considered as reaching the reaction equilibrium.

[0036] In Example 4, the time to reach equilibrium was 330 minutes, and the fructose conversion rate was 26.4%.

[0037] Example 5 The preparation of an immobilized isomerase includes the following steps: (1) Immobilized isomerase 2g of DAEase enzyme and 9g of carrier were dispersed in 25g of sodium phosphate buffer and stirred at room temperature for 1.1h. After centrifugation to remove the supernatant, 20g of glutaraldehyde solution was added and crosslinked at room temperature for 1.2h. The immobilized isomerase was obtained by freeze drying. The DAEase enzyme is derived from Bacillus thermophilus and has a molecular weight of 80,000 Da. The carrier is amino-porous silica microspheres with a particle size of 300 μm, a pore size of 80 nm, and a porosity of 65%. The sodium phosphate buffer solution has a concentration of 0.1 mol / L and a pH of 7. The concentration of the glutaraldehyde solution is 0.7 g / L.

[0038] (2) Catalytic preparation of allulose 1g of immobilized isomerase was added to 35g of a 500g / L fructose solution containing 1.5mmol / L manganese chloride. The mixture was stirred at 70℃, pH 7.5, and 350rpm until equilibrium was reached. The equilibrium state is when the concentrations of fructose and allulose in the mixture no longer change; The method for determining the equilibrium state is as follows: after the reaction has been going on for 1 hour, the concentrations of fructose and allulose in the solution are measured every 15 minutes until the concentration fluctuation is less than 5%, which is considered as reaching the reaction equilibrium.

[0039] In Example 5, the time to reach equilibrium was 330 minutes, and the fructose conversion rate was 26.1%.

[0040] Experimental Example 1 For the immobilized isomerases prepared in Examples 1-5, the activity of the immobilized glycosylated DAEase enzyme was measured by determining the amount of D-allulose produced. The reaction mixture (1 mL final volume) contained D-fructose (500 g / L), phosphate buffer (20 mM, pH 7.5), and Mn. 2+ The reaction was initiated by adding 0.03 g of immobilized isomerase (1.5 mmol / L). The reaction was incubated at 70°C for 1 h, and then stopped by placing the test tube in boiling water for 10 min. In the examples, the highest enzyme activity of the experimental subject in the same group of experiments was set as 100%, and the enzyme activity of the immobilized enzyme in the other examples was the percentage of the highest enzyme activity, which was the relative enzyme activity (%).

[0041] Table 1. Relative enzyme activities of immobilized isomerases

[0042] As shown in the table above, Example 4 omitted the step of preparing the lectin carrier based on Example 2. The relative enzyme activity of the prepared immobilized glycosylated DAEase enzyme was 84.1%, which was 15.9% lower than 100% in Example 2, showing a significant decrease in enzyme activity. This may be because the glycosylated DAEase enzyme was randomly adsorbed on the carrier surface in Example 4, and the active center was masked, resulting in a significant decrease in catalytic activity.

[0043] The relative enzyme activity of the immobilized isomerase in Example 5 was slightly lower than that in Example 4. In Example 5, the glycosylation step of DAEase enzyme was omitted from the model of Example 4, and DAEase enzyme was directly used for loading. This indicates that the glycosylation step did not cover or affect the active site, and did not have a negative impact on its catalytic activity.

[0044] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing allulose by catalyzing the conversion of fructose using an immobilized isomerase, characterized in that, The method includes the following steps: glycosylation of DAEase enzyme, preparation of lectin carrier, immobilization of glycosylated DAEase enzyme, and catalytic preparation of allulose. The glycosylation of the DAEase enzyme involves introducing a sugar chain onto the DAEase enzyme to obtain a glycosylated DAEase enzyme. The method for preparing the lectin carrier is as follows: aminated porous microspheres are dispersed in a lectin solution, incubated by rotation at 3-5°C for 22-24 hours, washed with phosphate buffer, and then freeze-dried to obtain the lectin carrier. The method for immobilizing glycosylated DAEase enzyme is as follows: glycosylated DAEase enzyme and lectin carrier are dispersed in sodium phosphate buffer, stirred at room temperature for 1-1.2 h, centrifuged to remove supernatant, added to glutaraldehyde solution, crosslinked at room temperature for 1-1.5 h, and freeze-dried to obtain immobilized isomerase.

2. The method for preparing allulose by catalyzing the conversion of fructose using an immobilized isomerase according to claim 1, characterized in that, The glycosylation method of the DAEase enzyme is as follows: DAEase enzyme and aldehyde-modified mannose are diluted with sodium phosphate buffer to prepare DAEase enzyme solution and aldehyde-modified mannose solution, respectively. The two solutions are mixed evenly, and after 6-8 min, 2-methylpyridineborane solution is added. The reaction is carried out in the dark at 3-5℃ for 4-5 h. After dialysis, glycosylated DAEase enzyme is obtained.

3. The method for preparing allulose by immobilized isomerase-catalyzed fructose conversion according to claim 1, characterized in that, In the glycosylation step of the DAEase enzyme, The molecular weight of the DAEase enzyme is 50,000-100,000 Da; The aldehyde-modified mannose is aldehyde-D-mannose; The 2-methylpyridineborane solution is obtained by dissolving 2-methylpyridineborane in sodium phosphate buffer.

4. The method for preparing allulose by catalyzing the conversion of fructose using an immobilized isomerase according to claim 3, characterized in that, In the glycosylation step of the DAEase enzyme, The concentration of the DAEase enzyme solution is 2-2.5 g / L; The concentration of the aldehyde-modified mannose solution is 2.5-3.5 g / L; The concentration of the 2-methylpyridineborane solution is 4-5 g / L. The mass ratio of the DAEase enzyme solution, the aldehyde-modified mannose solution, and the 2-methylpyridineborane solution is 10:1.2-1.4:4-6. The sodium phosphate buffer solution has a concentration of 0.08-0.12 mol / L and a pH of 6.8-7.

2.

5. The method for preparing allulose by fructose conversion catalyzed by immobilized isomerase according to claim 1, characterized in that, In the step of preparing the lectin carrier The lectin is concanavalin A; The concanavalin A solution is obtained by dissolving concanavalin A in sodium phosphate buffer solution, and the solution contains 18-22 mg / mL of concanavalin A. The mass ratio of the aminated microspheres to the canavanine A solution is 1:18-22.

6. The method for preparing allulose by immobilized isomerase-catalyzed fructose conversion according to claim 1, characterized in that, In the step of preparing the lectin carrier The aminated microspheres are amino-porous silica microspheres with a particle size of 200-500 μm, a pore size of 50-100 nm, and a porosity of 60-70%. The amino loading of the aminated microspheres is 2.8-3.2 mmol / g; The sodium phosphate buffer solution has a concentration of 0.08-0.12 mol / L and a pH of 6.8-7.

2.

7. The method for preparing allulose by immobilized isomerase-catalyzed fructose conversion according to claim 1, characterized in that, In the step of immobilizing glycosylated DAEase enzyme The mass ratio of the glycosylated DAEase enzyme, lectin carrier, sodium phosphate buffer, and glutaraldehyde solution is 0.15:4-5:10-15:8-12. The concentration of the glutaraldehyde solution is 0.6-0.8 g / L; The sodium phosphate buffer solution has a concentration of 0.08-0.12 mol / L and a pH of 6.8-7.

2.

8. The method for preparing allulose by fructose conversion catalyzed by immobilized isomerase according to claim 1, characterized in that, The method for catalytically preparing allulose involves adding an immobilized isomerase to a fructose solution containing 1-2 mmol / L manganese chloride, and stirring the reaction at 68-72°C and pH 7.4-7.6 until equilibrium is reached.

9. The method for preparing allulose by immobilized isomerase-catalyzed fructose conversion according to claim 8, characterized in that, The concentration of the fructose solution is 450-550 g / L, and the mass ratio of the immobilized isomerase to the fructose solution is 1:30-40.

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