A method for preparing d-allulose

Abstract

The application discloses a preparation method of D-allulose, which comprises the following steps: (1) preparing D-fructose into a solution, and then sequentially performing activated carbon decolorization and ion exchange to obtain a refined D-fructose solution; adding the refined D-fructose solution into a batching tank, then adding immobilized D-allulose 3-epimerase and a composite additive, and performing isomerization reaction to obtain a D-allulose crude solution; (2) performing decolorization, filtration, ion exchange, nanofiltration and chromatographic separation on the D-allulose crude solution, concentrating a D-allulose dilute solution separated out, and then performing crystallization or drying to prepare D-allulose. The magnetic immobilized D-allulose 3-epimerase prepared by a specific method, the composite additive compounded by specific raw materials, and the material circulation and separation process are systematically matched, so that the high efficiency, stability and economy of the whole process of D-allulose production are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosynthesis, and particularly relates to a preparation method of D-psicose. BACKGROUND

[0002] D-psicose is an important rare sugar, which exists in a small amount in cane molasses, dried fruits, sugar products, wheat and Rhus plants in nature. Its name originates from the fact that a small amount of D-psicose can also be separated from the antibiotic psicose adenosine. D-psicose is absorbed into the blood circulation through the small intestine in the human body, and is not metabolized into energy after being absorbed by the small intestine, and has a low fermentation utilization degree for intestinal microorganisms. D-psicose has a variety of important physiological functions: neuroprotective effect, blood glucose reduction, fat reduction, active oxygen cluster removal, antioxidant, cancer cell proliferation inhibition, low-calorie sweetener, etc.

[0003] The preparation process of D-psicose can be divided into chemical preparation and biological preparation. The chemical preparation method has not achieved corresponding results in practice due to a series of reasons such as complex purification product steps, serious chemical pollution and a large number of by-products. The biological preparation method is the main direction of the preparation of D-psicose, which has a single reaction and simple purification steps. At present, the main method for producing D-psicose is enzyme conversion, which uses D-fructose as a substrate to generate D-psicose under the catalysis of D-psicose-3-epimerase. D-psicose-3-epimerase has a reaction equilibrium constant, which is generally between 28-33%. D-fructose is converted from glucose under the catalysis of glucose isomerase. Glucose isomerase also has a reaction equilibrium constant, which is generally between 42-45%. In the case of using a single enzyme reaction to produce high-purity D-psicose, an additional purification process is needed to separate and remove high-concentration fructose from the reaction product. Therefore, the high raw material cost, the expensive product and by-product separation cost and the relatively low product yield limit its application.

[0004] Meanwhile, in the process of synthesizing D-psicose by using traditional biological enzyme method, free crude enzyme has the disadvantages of poor chemical stability, difficult collection after enzyme reaction, etc., which is not conducive to automatic production and has high production cost. Therefore, by using immobilized enzyme technology, D-psicose-3-epimerase is immobilized. Compared with free enzyme, immobilized enzyme not only maintains the characteristics of high efficiency and specificity of enzyme catalysis, but also greatly improves the thermal stability and chemical stability of the enzyme. For example, Chinese patent application CN115806966A discloses a D-psicose 3-epimerase immobilized enzyme and a preparation method and application thereof, which comprises an enzyme carrier and D-psicose 3-epimerase immobilized on the enzyme carrier. The enzyme carrier is a short-chain amino resin with a carbon atom number of 2-4, and the D-psicose 3-epimerase is from Rhizobium freirei. For another example, Chinese patent application CN116355888A discloses a D-psicose-3-epimerase immobilized enzyme preparation and a fixing method thereof. The fixing method is mainly embedding method using sodium alginate as carrier and adsorption method using single resin as carrier. D-psicose-3-epimerase is immobilized by embedding with sodium alginate and cross-linking with CaCl2. The enzyme activity recovery rate remains above 65% of the initial enzyme activity after 10 times of repeated use. Although the above-mentioned scheme solves the problems of poor chemical stability of free enzyme and difficult collection after reaction, etc., the above-mentioned scheme usually has the problems of large decrease of enzyme activity and reduction of immobilized enzyme amount, and the enzyme needs to be separated and purified in advance, which increases the immobilization cost. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a preparation method of D-psicose, which improves the loading amount and enzyme activity retention rate of D-psicose-3-epimerase on the immobilized carrier, and simultaneously adds a composite additive in the process of synthesizing D-psicose, so as to synergistically improve the conversion rate and yield of D-psicose.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0007] A preparation method of D-psicose, comprising the following steps:

[0008] (1) After preparing D-fructose into a solution, active carbon decolorization and ion exchange are sequentially performed to obtain a refined D-fructose solution; the refined D-fructose solution is added into a batching tank, and then immobilized D-psicose 3-epimerase and a composite additive are added for isomerization reaction. After the reaction is completed, the immobilized enzyme is recovered from the reaction solution by magnetic separation to obtain a D-psicose crude solution;

[0009] (2) The D-psicose crude solution obtained in step (1) is subjected to decolorization, filtration, ion exchange, nanofiltration, and chromatographic separation, and the separated D-fructose dilute solution is concentrated and used in step (1), and the D-psicose dilute solution is concentrated and subjected to crystallization or drying to obtain D-psicose.

[0010] Preferably, in step (1), the mass concentration of the refined D-fructose solution is 30-50%, the amount of the immobilized D-psicose 3-epimerase added is 5-8% of the mass of the refined D-fructose solution, and the amount of the composite additive added is 2-5% of the mass of the refined D-fructose solution.

[0011] Preferably, in step (1), the preparation method of the immobilized D-psicose 3-epimerase comprises the following steps:

[0012] S1, iron chloride and ferrous chloride are added to deionized water to obtain a mixed solution, sodium citrate is added to the mixed solution and stirred uniformly, then ammonia water is added under a nitrogen atmosphere, the pH is adjusted, aging is performed, the magnetic Fe3O4 particles are separated by a magnetic field after aging is completed, then the particles are washed and dried to obtain Fe3O4 particles; the Fe3O4 particles are added to an ethanol aqueous solution, followed by addition of cetyltrimethylammonium bromide, ammonia water, and triisopropylbenzene, the mixture is stirred uniformly, then tetraethyl orthosilicate is added, stirring reaction is performed, the product is collected by magnetic separation after the reaction is completed, and the product is washed, dried, and calcined to obtain an immobilized carrier;

[0013] S2, the immobilized carrier in step S1 is added to an ethanol aqueous solution, followed by addition of γ-glycidoxypropyltrimethoxysilane, and heating reaction is performed, then the product is filtered, washed, and dried to obtain a pretreated immobilized carrier;

[0014] S3, the pretreated immobilized carrier in step S2 is added to a borate buffer solution, followed by addition of L-lysine, and constant-temperature reaction is performed, then the product is filtered, washed, and dried to obtain a modified immobilized carrier;

[0015] S4, the modified immobilized carrier in step S3 is added to a MES buffer solution, followed by addition of EDC and NHS, and stirring activation is performed, then the product is separated by a magnetic field after activation is completed, and the product is washed to obtain an activated immobilized carrier; the activated immobilized carrier is added to a PBS buffer solution containing D-psicose 3-epimerase, and immobilization is performed, then the product is separated by a magnetic field after immobilization is completed, and the product is repeatedly washed with a buffer solution until no enzyme activity is detected in the washing solution, thereby obtaining the immobilized D-psicose 3-epimerase.

[0016] Preferably, the concentration of ferric chloride in the mixed solution in step S1 is 0.3-0.8 mol / L, the concentration of ferrous chloride is 0.2-0.4 mol / L, the mass concentration of the ammonia water is 25-28%, the molar ratio of sodium citrate to ferric chloride is 0.3-0.4:1, the pH is 10-11, the aging temperature is 70-80℃, and the time is 1-2 h.

[0017] Preferably, the mass ratio of ethanol to water in the aqueous ethanol solution in step S1 is 8-9:1-2, the mass ratio of Fe3O4 particles, cetyltrimethylammonium bromide, ammonia water, triisopropylbenzene, and tetraethyl orthosilicate is 90-100:60-90:300-400:30-50:150-200, the temperature of the stirring reaction is 40-60℃, the time is 5-8 h, and the calcination process is as follows: under a nitrogen atmosphere, the temperature is raised to 450-500℃ at a temperature raising rate of 2-3℃ / min, and the temperature is kept for 2-3 h.

[0018] In the present application, Fe3O4 particles are prepared by a coprecipitation method as magnetic cores, and sodium citrate is added during the preparation process, which is adsorbed on the surface of nascent Fe3O4 particles, effectively preventing the agglomeration of the particles during the aging process through its electrostatic repulsion and steric hindrance effect, thereby ensuring the dispersibility of the magnetic cores; meanwhile, the citrate wrapped on the surface of the Fe3O4 particles has a large number of negative charges, which is conducive to the subsequent adsorption of positively charged cetyltrimethylammonium bromide on the surface, laying a good foundation for the subsequent coating of silica; then, using cetyltrimethylammonium bromide as a template agent and triisopropylbenzene as a pore-expanding agent, a uniform mesoporous silica shell layer is coated on the surface of the magnetic core by hydrolysis of tetraethyl orthosilicate, providing a suitable carrier for subsequent functionalization and enzyme immobilization.

[0019] Preferably, the mass ratio of the immobilized carrier to γ-glycidoxypropyltrimethoxysilane in step S2 is 90-100:8-13, the temperature of the heating reaction is 55-65℃, and the time is 2-3 h.

[0020] In the present application, γ-glycidoxypropyltrimethoxysilane is reacted with the immobilized carrier to introduce epoxy groups on the immobilized carrier, which is conducive to the subsequent functionalization of the carrier.

[0021] Preferably, the pH of the borate buffer in step S3 is 9-10, the mass ratio of the pretreated immobilized carrier to L-lysine is 100:5-9, the temperature of the constant-temperature reaction is 40-60℃, and the time is 8-12 h.

[0022] In the present application, L-lysine is a natural amino acid, which has a flexible methylene side chain in its molecular structure, and has an α-amino group and an ε-amino group at the same time, and the amino group is reacted with the epoxy group introduced in the previous step to covalently connect the L-lysine molecule to the carrier; L-lysine plays a flexible spacing role, solving the problem that the enzyme is directly fixed on the surface of a rigid carrier, which severely limits the conformational flexibility of the enzyme, thereby causing a large decrease in the activity of the enzyme. By introducing L-lysine, the enzyme is fixed at the end of L-lysine, obtaining a larger activity radius and a more free conformational change space, thereby significantly relieving the steric hindrance effect, so that the immobilized enzyme retains higher catalytic activity, and the carboxyl group at the end of L-lysine provides a site for the next reaction.

[0023] Preferably, the pH of the MES buffer in step S4 is 5-6, the mass ratio of the modified immobilized carrier, EDC and NHS is 100:15-30:10-25, the temperature of the stirring activation is 2-6℃, and the time is 1-2h; the pH of the PBS buffer is 7-8, the mass concentration of D- Psicose 3-epimerase is 3-5g / L, and the immobilization temperature is 4-6℃, and the time is 10-15h.

[0024] In the present application, the carboxyl group at the end of the L-lysine arm on the modified immobilized carrier is activated under low-temperature acidic conditions (MES buffer) to convert it into a highly active NHS ester, and then the primary amino group (mainly from lysine residues) on the surface of D- Psicose 3-epimerase is reacted with the NHS ester to form a stable amide bond, thereby firmly fixing the enzyme at the end of the spacer arm. The entire reaction can be carried out in an aqueous environment close to neutral, causing minimal damage to the activity of the enzyme, and the amide bond formed is chemically very stable and can withstand repeated flushing and pH changes under industrial conditions, thereby ensuring that the enzyme does not leak (fall off) from the carrier in large quantities, allowing the immobilized enzyme to be used for a long time and repeatedly.

[0025] Preferably, the preparation method of the composite additive in step (1) is as follows:

[0026] According to weight parts, 30-50 parts of glycerol, 8-13 parts of betaine, 0.5-1 part of manganese sulfate, 12-17 parts of L-arginine are added into 1000 parts of deionized water, and after stirring uniformly, it is obtained.

[0027] In the present application, the prepared composite additive is scientifically compounded with four components of manganese sulfate, glycerol, betaine and L-arginine, manganese sulfate provides Mn 2+As a cofactor of enzyme, it can be combined with active center efficiently, induce enzyme protein to form catalytic conformation with highest activity, and catalyze the activity of enzyme; at higher temperature (55-65℃), D-psicose 3-epimerase has a tendency of spontaneous unfolding, and glycerol, as a polyol, can provide basic thermodynamic stability by increasing the viscosity of the system, reducing water activity and forming hydrogen bonds with the surface of the enzyme; betaine can stabilize the overall three-dimensional conformation of the enzyme, reduce the disturbance of the microenvironment around the active center, and ensure that the metal ion plays a role in a more stable and ideal geometric configuration, thereby prolonging its efficient catalytic life; the synergistic effect of the two greatly improves the stability of the enzyme; L-arginine, as an excellent protein aggregation inhibitor, its molecules can effectively interact with the hydrophobic patches on the surface of the enzyme, prevent harmful contact between enzyme molecules, improve the reuse rate of immobilized enzyme, and significantly prolong the service life of immobilized enzyme.

[0028] Preferably, the temperature of the isomerization reaction in step (1) is 55-65℃, and the time is 15-20h.

[0029] Preferably, the decolorization process in step (2) is: pumping the D-psicose crude liquid into a decolorization column filled with granular activated carbon, treating at a temperature of 40-60℃ and a flow rate of 2-4 BV / h; the filtration process is: sequentially passing the decolorized liquid through a precision filter and a 0.22μm microporous filter membrane; the ion exchange process is: sequentially passing the clarified liquid through a strong acid cation exchange resin column (H + type) and a weak base anion exchange resin column (OH - type) in parallel, controlling the temperature at 20-30℃ and the flow rate at 2-5 BV / h; the nanofiltration process is: passing the ion-exchanged liquid through a 150Da nanofiltration membrane at 0.5-2MPa to obtain a retentate; the chromatographic separation process is: using calcium ion (Ca 2+ ) type strong acid cation exchange resin as the stationary phase and deionized water as the mobile phase, and controlling the chromatographic separation temperature at 50-65℃.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] (1) The D-psicose preparation method provided by the application realizes high efficiency, stability and economy of the whole process of D-psicose production by systematically matching the magnetic immobilized D-psicose 3-epimerase prepared by a specific method, the composite additive compounded by specific raw materials, and the material circulation and separation process. The method of the application not only significantly improves the operation stability and catalytic performance of the core catalyst (D-psicose 3-epimerase) through advanced immobilization technology and microenvironment regulation strategy, but also ensures high purity of the product while recycling and recycling the unconverted substrate D-fructose through subsequent decolorization, filtration, ion exchange, nanofiltration, chromatographic separation and other technologies, solves the key technical bottlenecks such as short enzyme life, difficult product separation and low raw material utilization rate in traditional biological catalysis method, and has good industrialization implementation value.

[0032] (2) The D-psicose preparation method provided by the application successfully constructs a flexible covalent connection between the inorganic carrier and the enzyme protein by constructing a mesoporous SiO2 shell on the surface of Fe3O4 magnetic core and then functionally modifying it with gamma-glycidoxypropyltrimethoxysilane and L-lysine. This structure not only gives the catalyst excellent magnetic separation performance and high enzyme loading potential, but more importantly, the introduced L-lysine as a flexible spacer reduces the steric hindrance of D-psicose 3-epimerase on the immobilized carrier, effectively alleviating the constraint of the carrier surface on the three-dimensional conformation of the enzyme protein, so that the immobilized enzyme retains higher catalytic activity. At the same time, the stable amide bond covalently connected by EDC / NHS chemistry significantly reduces the enzyme shedding during operation, thereby obtaining a high-performance immobilized enzyme preparation with high activity, high stability and excellent reusability.

[0033] (3) The D-psicose preparation method provided by the application optimizes the microenvironment of the isomerization reaction by scientifically compounding manganese sulfate, glycerol, betaine and L-arginine. Manganese sulfate as a specific activator directly acts on the enzyme active center to improve the catalytic rate. Glycerol and betaine as thermodynamic stabilizers, together inhibit the enzyme protein unfolding caused by high temperature. L-arginine as a physical conformation stabilizer effectively inhibits the aggregation and inactivation of enzyme molecules. Through the synergistic effect of the four substances, the problem of enzyme deactivation in industrial application is fundamentally solved. Compared with traditional single additives, the service life and batch circulation ability of the immobilized enzyme are greatly prolonged. DETAILED DESCRIPTION

[0034] The technical solutions of the present application will be described clearly and completely below in connection with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application.

[0035] Unless otherwise specified, the chemicals and materials in the present application are purchased through market channels or synthesized from raw materials purchased through market channels.

[0036] Embodiment 1

[0037] A preparation method of D-psicose, comprising the following steps:

[0038] (1) D-fructose is prepared into a solution, and then subjected to activated carbon decolorization and ion exchange in sequence to obtain a refined D-fructose solution (mass concentration of 40%); the refined D-fructose solution is added into a batching tank, and then immobilized D-psicose 3-epimerase and a composite additive are added, the addition amount of the immobilized D-psicose 3-epimerase is 7% of the mass of the refined D-fructose solution, and the addition amount of the composite additive is 3% of the mass of the refined D-fructose solution, and isomerization reaction is carried out at 55-65°C for 20h, after the reaction is completed, the immobilized enzyme is recovered from the reaction solution by magnetic separation to obtain a D-psicose crude solution;

[0039] (2) The D-psicose crude solution obtained in step (1) is subjected to decolorization, filtration, ion exchange, nanofiltration and chromatographic separation, the separated D-fructose dilute solution is concentrated and then used in step (1), and the D-psicose dilute solution is concentrated, crystallized or dried to prepare D-psicose.

[0040] In step (1), the preparation method of the immobilized D-psicose 3-epimerase comprises the following steps:

[0041] S1, iron chloride and ferrous chloride were added into deionized water to obtain a mixed solution (the concentration of iron chloride was 0.5 mol / L, and the concentration of ferrous chloride was 0.3 mol / L), sodium citrate was added into the mixed solution, the molar ratio of sodium citrate to iron chloride was 0.35:1, and then the mixture was stirred uniformly, 25% ammonia water was added under a nitrogen atmosphere, the pH was adjusted to 11, and the mixture was aged at 75℃ for 1.5 h, then the magnetic Fe3O4 particles were separated by a magnetic field, and then the particles were washed and dried to obtain Fe3O4 particles; 95 g of the Fe3O4 particles were added into an ethanol aqueous solution (the mass ratio of ethanol to water was 8.5:1.5), then 80 g of hexadecyl trimethyl ammonium bromide, 350 g of 20% ammonia water, and 40 g of triisopropylbenzene were added, the mixture was stirred uniformly, then 180 g of tetraethyl orthosilicate was added, the mixture was stirred at 50℃ for 7 h, the product was collected by magnetic separation after the reaction was completed, and the product was washed, dried, and calcined, the calcination process was as follows: the temperature was increased to 480℃ at a temperature increasing rate of 2.5℃ / min under a nitrogen atmosphere, and the temperature was kept for 2.5 h, to obtain the immobilized carrier;

[0042] S2, 95 g of the immobilized carrier in step S1 was added into 1.5 L of an ethanol aqueous solution (the mass fraction of ethanol was 75%), then 11 g of γ-glycidoxypropyltrimethoxysilane was added, and the mixture was reacted at 60℃ for 2.5 h, then the mixture was filtered, washed, and dried to obtain a pretreated immobilized carrier;

[0043] S3, 95 g of the pretreated immobilized carrier in step S2 was added into 1.5 L of a borate buffer solution with a pH of 9.5, then 7 g of L-lysine was added, and the mixture was reacted at 50℃ for 10 h, then the mixture was filtered, washed, and dried to obtain a modified immobilized carrier;

[0044] S4, 100 g of the modified immobilized carrier in step S3 was added into 1.5 L of a MES buffer solution with a pH of 5.5, then 25 g of EDC and 20 g of NHS were added, the mixture was stirred at 4℃ for 1.5 h, then the mixture was separated by a magnetic field after the activation was completed, and the activated immobilized carrier was obtained after washing; 100 g of the activated immobilized carrier was added into 2 L of a PBS buffer solution containing D-psicose 3-epimerase (the pH of the PBS buffer solution was 7.5, and the mass concentration of D-psicose 3-epimerase was 4 g / L), and the mixture was immobilized at 5℃ for 13 h, then the immobilized D-psicose 3-epimerase was separated by a magnetic field, and the immobilized D-psicose 3-epimerase was obtained after repeated washing with the buffer solution until no enzyme activity was detected in the washing solution.

[0045] The preparation method of the composite additive is as follows:

[0046] 40 parts of glycerol, 11 parts of betaine, 0.8 parts of manganese sulfate, 15 parts of L-arginine are added into 1000 parts of deionized water, and after stirring uniformly, it is obtained.

[0047] The decoloring process in step (2) is that the D-allulose crude solution is pumped into a decoloring column filled with granular activated carbon, and is treated at a temperature of 50℃ and a flow rate of 3 BV / h; the filtering process is that the decolored solution is sequentially passed through a precision filter and a 0.22 μm microporous filter membrane; the ion exchange process is that the clarified solution is sequentially passed through a strong acid cation exchange resin column (H + +) type and a weak base anion exchange resin column (OH - +) type in parallel, with temperature control at 25℃ and flow rate control at 4 BV / h; the nanofiltration process is that the ion-exchanged solution is passed through a 150 Da nanofiltration membrane at 1 MPa to obtain a retentate; the chromatographic separation process is that a strong acid cation exchange resin of calcium ion (Ca 2+ +) type is used as the stationary phase, and deionized water is used as the mobile phase, with chromatographic separation temperature control at 60℃.

[0048] Example 2

[0049] A preparation method of D-allulose, comprising the following steps:

[0050] (1) D-fructose is prepared into a solution, and then is sequentially subjected to activated carbon decoloring and ion exchange to obtain a refined D-fructose solution (mass concentration of 40%); the refined D-fructose solution is added into a batching tank, and then fixed D-allulose 3-epimerase and a composite additive are added, the addition amount of the fixed D-allulose 3-epimerase is 5% of the mass of the refined D-fructose solution, and the addition amount of the composite additive is 2% of the mass of the refined D-fructose solution, isomerization is carried out at 60℃ for 20 h, and after the reaction is completed, the fixed enzyme is recovered from the reaction solution by magnetic separation to obtain a D-allulose crude solution;

[0051] (2) The D-allulose crude solution obtained in step (1) is subjected to decoloring, filtering, ion exchange, nanofiltration and chromatographic separation, the separated D-fructose dilute solution is concentrated and then used in step (1), and the D-allulose dilute solution is concentrated and then crystallized or dried to prepare D-allulose.

[0052] In step (1), the preparation method of the fixed D-allulose 3-epimerase comprises the following steps:

[0053] S1, iron chloride, ferrous chloride were added into deionized water, and stirred to obtain a mixed solution (the concentration of iron chloride was 0.3 mol / L, and the concentration of ferrous chloride was 0.2 mol / L), then sodium citrate was added into the mixed solution, the molar ratio of sodium citrate to iron chloride was 0.3:1, and stirred uniformly, then 25% ammonia water was added under nitrogen atmosphere, the pH was adjusted to 10, and the mixture was aged at 70℃ for 2h, then the magnetic Fe3O4 particles were separated by magnetic field, and then washed and dried to obtain Fe3O4 particles; 90g of Fe3O4 particles were added into an ethanol aqueous solution (the mass ratio of ethanol to water was 8:2), then 60g of hexadecyl trimethyl ammonium bromide, 300g of 20% ammonia water, and 30g of triisopropylbenzene were added, the mixture was stirred uniformly, then 150g of tetraethyl orthosilicate was added, the mixture was stirred at 40℃ for 8h, then the product was collected by magnetic separation, and then washed, dried, and calcined, the calcination process was as follows: under nitrogen atmosphere, the temperature was increased to 450℃ at a rate of 2℃ / min, and kept for 3h, to obtain the immobilized carrier;

[0054] S2, 90g of the immobilized carrier in step S1 was added into 1.5L of an ethanol aqueous solution (the mass fraction of ethanol was 75%), then 8g of γ-glycidoxypropyltrimethoxysilane was added, and the mixture was reacted at 55℃ for 3h, then the product was filtered, washed, and dried to obtain the pretreated immobilized carrier;

[0055] S3, 90g of the pretreated immobilized carrier in step S2 was added into 1.5L of a borate buffer solution with pH of 9, then 5g of L-lysine was added, and the mixture was reacted at 40℃ for 12h, then the product was filtered, washed, and dried to obtain the modified immobilized carrier;

[0056] S4, 100g of the modified immobilized carrier in step S3 was added into 1.5L of a MES buffer solution with pH of 5, then 15g of EDC and 10g of NHS were added, and the mixture was stirred at 2℃ for 2h, then the product was separated by magnetic separation, and washed to obtain the activated immobilized carrier; 100g of the activated immobilized carrier was added into 2L of a PBS buffer solution containing D-psicose 3-epimerase (the pH of the PBS buffer solution was 7, and the mass concentration of D-psicose 3-epimerase was 3g / L), and the mixture was immobilized at 4℃ for 15h, then the product was separated by magnetic field, and repeatedly washed with the buffer solution until no enzyme activity was detected in the washing solution, to obtain the immobilized D-psicose 3-epimerase.

[0057] The preparation method of the composite additive is as follows:

[0058] According to weight parts, 30 parts of glycerol, 8 parts of betaine, 0.5 parts of manganese sulfate, and 12 parts of L-arginine were added into 1000 parts of deionized water, and the mixture was stirred uniformly to obtain the composite additive.

[0059] The decoloring process in step (2) is: pumping the D-psicose crude solution into a decoloring column filled with granular activated carbon, treating at a temperature of 50℃ and a flow rate of 3 BV / h; the filtering process is: sequentially passing the decolored solution through a precision filter and a 0.22 μm microporous filter membrane; the ion exchange process is: sequentially passing the clarified solution through a strong acid cation exchange resin column (H + type) and a weak base anion exchange resin column (OH - type) in parallel, controlling the temperature at 25℃ and the flow rate at 4 BV / h; the nanofiltration process is: passing the ion-exchanged solution through a 150 Da nanofiltration membrane at 1 MPa to obtain a retentate; the chromatographic separation process is: using calcium ion (Ca 2+ ) type strong acid cation exchange resin as the stationary phase and deionized water as the mobile phase, and controlling the chromatographic separation temperature at 60℃.

[0060] Example 3

[0061] A method for preparing D-psicose, comprising the following steps:

[0062] (1) preparing a D-fructose solution, then sequentially performing activated carbon decoloring and ion exchange to obtain a refined D-fructose solution (mass concentration of 40%); adding the refined D-fructose solution into a batching tank, then adding immobilized D-psicose 3-epimerase and a composite additive, the amount of the immobilized D-psicose 3-epimerase added is 8% of the mass of the refined D-fructose solution, and the amount of the composite additive added is 5% of the mass of the refined D-fructose solution, isomerizing at 60℃ for 20 h, recovering the immobilized enzyme from the reaction solution by magnetic separation after the reaction is completed, and obtaining a D-psicose crude solution;

[0063] (2) subjecting the D-psicose crude solution obtained in step (1) to decoloring, filtering, ion exchange, nanofiltration and chromatographic separation, concentrating the separated D-fructose dilute solution, and using it in step (1), and concentrating the D-psicose dilute solution and then crystallizing or drying to prepare D-psicose.

[0064] In step (1), the method for preparing the immobilized D-psicose 3-epimerase comprises the following steps:

[0065] S1, iron chloride, ferrous chloride were added into deionized water, and stirred to obtain a mixed solution (the concentration of iron chloride was 0.8 mol / L, and the concentration of ferrous chloride was 0.4 mol / L), then sodium citrate was added into the mixed solution, the molar ratio of sodium citrate to iron chloride was 0.4:1, and stirred uniformly, then 28% ammonia water was added under nitrogen atmosphere, the pH was adjusted to 11, and the mixture was aged at 80℃ for 1h, then the magnetic Fe3O4 particles were separated by magnetic field, and then washed and dried to obtain Fe3O4 particles; 100g of Fe3O4 particles were added into an ethanol aqueous solution (the mass ratio of ethanol to water was 9:1), then 90g of hexadecyl trimethyl ammonium bromide, 400g of 20% ammonia water, and 50g of triisopropylbenzene were added, the mixture was stirred uniformly, then 200g of tetraethyl orthosilicate was added, the mixture was stirred at 60℃ for 5h, the product was collected by magnetic separation after the reaction was completed, and then washed, dried, and calcined, the calcination process was as follows: under nitrogen atmosphere, the temperature was raised to 500℃ at a temperature raising rate of 3℃ / min, and the mixture was kept at 500℃ for 2h, to obtain the immobilized carrier;

[0066] S2, 100g of the immobilized carrier in step S1 was added into 1.5L of an ethanol aqueous solution (the mass fraction of ethanol was 75%), then 13g of γ-glycidoxypropyltrimethoxysilane was added, and the mixture was reacted at 65℃ for 2h, then the mixture was filtered, washed, and dried to obtain the pretreated immobilized carrier;

[0067] S3, 100g of the pretreated immobilized carrier in step S2 was added into 1.5L of a borate buffer solution with pH of 10, then 9g of L-lysine was added, and the mixture was reacted at 0℃ for 8h, then the mixture was filtered, washed, and dried to obtain the modified immobilized carrier;

[0068] S4, 100g of the modified immobilized carrier in step S3 was added into 1.5L of a MES buffer solution with pH of 6, then 30g of EDC and 25g of NHS were added, and the mixture was stirred at 6℃ for 1h, then the mixture was separated by magnetic separation after the activation was completed, and then washed to obtain the activated immobilized carrier; 100g of the activated immobilized carrier was added into 2L of a PBS buffer solution containing D-psicose 3-epimerase (the pH of the PBS buffer solution was 8, and the mass concentration of D-psicose 3-epimerase was 5g / L), and the mixture was immobilized at 6℃ for 10h, then the mixture was separated by magnetic field after the immobilization was completed, and then repeatedly washed with the buffer solution until no enzyme activity was detected in the washing solution, to obtain the immobilized D-psicose 3-epimerase.

[0069] The preparation method of the composite additive is as follows:

[0070] According to weight parts, 50 parts of glycerol, 13 parts of betaine, 1 part of manganese sulfate, and 17 parts of L-arginine were added into 1000 parts of deionized water, and the mixture was stirred uniformly, to obtain the composite additive.

[0071] The decoloring process in step (2) is: pumping the D-psicose crude solution into a decoloring column filled with granular activated carbon, treating at a temperature of 50℃ and a flow rate of 3 BV / h; the filtering process is: sequentially passing the decolored solution through a precision filter and a 0.22 μm microporous filter membrane; the ion exchange process is: sequentially passing the clarified solution through a strong acid cation exchange resin column (H + type) and a weak base anion exchange resin column (OH - type) in parallel, controlling the temperature at 25℃ and the flow rate at 4 BV / h; the nanofiltration process is: passing the ion-exchanged solution through a 150 Da nanofiltration membrane at 1 MPa to obtain a retentate; the chromatographic separation process is: using calcium ion (Ca 2+ ) type strong acid cation exchange resin as the stationary phase and deionized water as the mobile phase, and controlling the chromatographic separation temperature at 60℃.

[0072] Comparative Example 1

[0073] A method for preparing D-psicose, characterized in that it comprises the following steps:

[0074] (1) After preparing a D-fructose solution, sequentially performing activated carbon decoloring and ion exchange to obtain a refined D-fructose solution (mass concentration of 40%); adding the refined D-fructose solution into a batching tank, then adding immobilized D-psicose 3-epimerase and a composite additive, the amount of immobilized D-psicose 3-epimerase added is 7% of the mass of the refined D-fructose solution, and the amount of the composite additive added is 3% of the mass of the refined D-fructose solution, isomerizing at 55-65℃ for 20 h, and then recovering the immobilized enzyme from the reaction solution by magnetic separation to obtain a D-psicose crude solution;

[0075] (2) Decoloring, filtering, ion exchanging, nanofiltrating and chromatographically separating the D-psicose crude solution obtained in step (1), and then concentrating the separated D-fructose dilute solution for use in step (1), and crystallizing or drying the D-psicose dilute solution to prepare D-psicose.

[0076] In step (1), the method for preparing the immobilized D-psicose 3-epimerase comprises the following steps:

[0077] S1, iron chloride and ferrous chloride were added into deionized water to obtain a mixed solution (the concentration of iron chloride was 0.5 mol / L, and the concentration of ferrous chloride was 0.3 mol / L), sodium citrate was added into the mixed solution, the molar ratio of sodium citrate to iron chloride was 0.35:1, and then the mixture was stirred uniformly, 25% ammonia water was added under a nitrogen atmosphere, the pH was adjusted to 11, and the mixture was aged at 75℃ for 1.5 h, then the magnetic Fe3O4 particles were separated by a magnetic field, and then the particles were washed and dried to obtain Fe3O4 particles; 95 g of the Fe3O4 particles were added into an ethanol aqueous solution (the mass ratio of ethanol to water was 8.5:1.5), then 80 g of hexadecyl trimethyl ammonium bromide, 350 g of 20% ammonia water, and 40 g of triisopropylbenzene were added, the mixture was stirred uniformly, then 180 g of tetraethyl orthosilicate was added, the mixture was stirred at 50℃ for 7 h, the product was collected by magnetic separation after the reaction was completed, and the product was washed, dried, and calcined, the calcination process was as follows: the temperature was increased to 480℃ at a temperature increasing rate of 2.5℃ / min under a nitrogen atmosphere, and the temperature was kept for 2.5 h, to obtain the immobilized carrier;

[0078] S2, 95 g of the immobilized carrier in step S1 was added into 1.5 L of an ethanol aqueous solution (the mass fraction of ethanol was 75%), then 11 g of γ-glycidoxypropyltrimethoxysilane was added, and the mixture was reacted at 60℃ for 2.5 h, and then the mixture was filtered, washed, and dried to obtain a pretreated immobilized carrier;

[0079] S3, 100 g of the pretreated immobilized carrier in step S2 was added into 1.5 L of a MES buffer solution with a pH of 5.5, then 25 g of EDC and 20 g of NHS were added, the mixture was stirred and activated at 4℃ for 1.5 h, the activated immobilized carrier was separated by a magnetic field after the activation was completed, and then the activated immobilized carrier was washed to obtain an activated immobilized carrier; 100 g of the activated immobilized carrier was added into 2 L of a PBS buffer solution containing D-alloketose 3-epimerase (the pH of the PBS buffer solution was 7.5, and the mass concentration of D-alloketose 3-epimerase was 4 g / L), and the mixture was immobilized at 5℃ for 13 h, the immobilized carrier was separated by a magnetic field after the immobilization was completed, and the immobilized carrier was repeatedly washed with the buffer solution until no enzyme activity was detected in the washing solution, to obtain the immobilized D-alloketose 3-epimerase.

[0080] The preparation method of the composite additive is as follows:

[0081] 40 parts of glycerol, 11 parts of betaine, 0.8 parts of manganese sulfate, and 15 parts of L-arginine were added into 1000 parts of deionized water, and the mixture was stirred uniformly to obtain the composite additive.

[0082] The decoloring process in step (2) is: pumping the D-psicose crude solution into a decoloring column filled with granular activated carbon, treating at a temperature of 50°C and a flow rate of 3 BV / h; the filtering process is: sequentially passing the decolored solution through a precision filter and a 0.22 μm microporous filter membrane; the ion exchange process is: sequentially passing the clarified solution through a strong acid cation exchange resin column (H + type) and a weak base anion exchange resin column (OH - type) in parallel, controlling the temperature at 25°C and the flow rate at 4 BV / h; the nanofiltration process is: passing the ion-exchanged solution through a 150 Da nanofiltration membrane at 1 MPa to obtain a retentate; the chromatographic separation process is: using calcium ion (Ca 2+ ) type strong acid cation exchange resin as the stationary phase and deionized water as the mobile phase, and controlling the chromatographic separation temperature at 60°C.

[0083] Compared with Example 1, no L-lysine is introduced into the immobilized D-psicose 3-epimerase of the present comparative example.

[0084] Comparative Example 2

[0085] A method for preparing D-psicose, characterized in that it comprises the following steps:

[0086] (1) preparing a D-fructose solution, then sequentially performing activated carbon decoloring and ion exchange to obtain a refined D-fructose solution (mass concentration of 40%); adding the refined D-fructose solution into a batching tank, then adding immobilized D-psicose 3-epimerase and a composite additive, the amount of the immobilized D-psicose 3-epimerase added is 7% of the mass of the refined D-fructose solution, and the amount of the composite additive added is 3% of the mass of the refined D-fructose solution, isomerizing at 55-65°C for 20 h, recovering the immobilized enzyme from the reaction solution by magnetic separation after the reaction is completed, and obtaining a D-psicose crude solution;

[0087] (2) subjecting the D-psicose crude solution obtained in step (1) to decoloring, filtering, ion exchange, nanofiltration and chromatographic separation, concentrating the separated D-fructose dilute solution, and using it in step (1), and concentrating the D-psicose dilute solution and then crystallizing or drying to prepare D-psicose.

[0088] In step (1), the method for preparing the immobilized D-psicose 3-epimerase comprises the following steps:

[0089] S1, iron chloride, ferrous chloride were added into deionized water, and stirred to obtain a mixed solution (the concentration of iron chloride was 0.5 mol / L, and the concentration of ferrous chloride was 0.3 mol / L), sodium citrate was added into the mixed solution, the molar ratio of sodium citrate to iron chloride was 0.35:1, and stirred uniformly, then 25% ammonia water was added under nitrogen atmosphere, the pH was adjusted to 11, and aged at 75℃ for 1.5h, after aging, the magnetic Fe3O4 particles were separated by magnetic field, then washed, dried, and Fe3O4 particles were obtained; 95g Fe3O4 particles were added into an ethanol aqueous solution (the mass ratio of ethanol to water was 8.5:1.5), then 80g hexadecyl trimethyl ammonium bromide, 350g 20% ammonia water, and 40g triisopropylbenzene were added, stirred uniformly, then 180g tetraethyl orthosilicate was added, and stirred at 50℃ for 7h, after reaction, the product was collected by magnetic separation, washed, dried, calcined, the calcination process was as follows: under nitrogen atmosphere, the temperature was increased to 480℃ at a heating rate of 2.5℃ / min, and kept for 2.5h, and the immobilized carrier was obtained;

[0090] S2, 100g immobilized carrier in step S1 was added into 2L PBS buffer containing D-psicose 3-epimerase (the pH of PBS buffer was 7.5, and the mass concentration of D-psicose 3-epimerase was 4g / L), and immobilized at 5℃ for 13h, after immobilization, the immobilized D-psicose 3-epimerase was separated by magnetic field, and repeatedly washed with buffer until no enzyme activity was detected in the washing liquid.

[0091] The preparation method of the composite additive was as follows:

[0092] According to weight parts, 40 parts of glycerol, 11 parts of betaine, 0.8 parts of manganese sulfate, and 15 parts of L-arginine were added into 1000 parts of deionized water, and stirred uniformly, and then the composite additive was obtained.

[0093] The decolorization process in step (2) was as follows: the D-psicose crude liquid was pumped into a decolorization column filled with granular activated carbon, and treated at a flow rate of 3 BV / h at a temperature of 50℃; the filtration process was as follows: the decolorized liquid was sequentially passed through a precision filter and a 0.22μm microporous filter membrane; the ion exchange process was as follows: the clarified liquid was sequentially passed through a strong acid cation exchange resin column (H + type) and a weak base anion exchange resin column (OH - type) in parallel, the temperature was controlled at 25℃, and the flow rate was controlled at 4BV / h; the nanofiltration process was as follows: the liquid after ion exchange was passed through a 150Da nanofiltration membrane at 1MPa, and a retentate was obtained; the chromatographic separation process was as follows: calcium ions (Ca 2+The temperature of the chromatographic separation is controlled at 60°C.

[0094] In comparison with Example 1, the immobilized D-psicose 3-epimerase in the present comparative example introduces D-psicose 3-epimerase onto the immobilized carrier by physical adsorption.

[0095] Comparative Example 3

[0096] A method for preparing D-psicose, characterized in that it comprises the following steps:

[0097] (1) D-fructose is prepared into a solution, and then subjected to decolorization with activated carbon and ion exchange in sequence to obtain a refined D-fructose solution (mass concentration of 40%); the refined D-fructose solution is added into a batching tank, followed by addition of immobilized D-psicose 3-epimerase and a composite additive, the addition amount of the immobilized D-psicose 3-epimerase being 7% of the mass of the refined D-fructose solution, and the addition amount of the composite additive being 3% of the mass of the refined D-fructose solution; isomerization reaction is carried out at 55-65°C for 20h, after which the immobilized enzyme is recovered from the reaction solution by magnetic separation to obtain a D-psicose crude solution;

[0098] (2) The D-psicose crude solution obtained in step (1) is subjected to decolorization, filtration, ion exchange, nanofiltration and chromatographic separation; the separated D-fructose dilute solution is concentrated and then used in step (1); and the D-psicose dilute solution is concentrated and then subjected to crystallization or drying to prepare D-psicose.

[0099] In step (1), the method for preparing the immobilized D-psicose 3-epimerase comprises the following steps:

[0100] S1, iron chloride and ferrous chloride were added into deionized water to obtain a mixed solution (the concentration of iron chloride was 0.5 mol / L, and the concentration of ferrous chloride was 0.3 mol / L), sodium citrate was added into the mixed solution, the molar ratio of sodium citrate to iron chloride was 0.35:1, and then the mixture was stirred uniformly, 25% ammonia water was added under a nitrogen atmosphere, the pH was adjusted to 11, and the mixture was aged at 75℃ for 1.5 h, then the magnetic Fe3O4 particles were separated by a magnetic field, and then the particles were washed and dried to obtain Fe3O4 particles; 95 g of the Fe3O4 particles were added into an ethanol aqueous solution (the mass ratio of ethanol to water was 8.5:1.5), then 80 g of hexadecyl trimethyl ammonium bromide, 350 g of 20% ammonia water, and 40 g of triisopropylbenzene were added, the mixture was stirred uniformly, then 180 g of tetraethyl orthosilicate was added, the mixture was stirred at 50℃ for 7 h, the product was collected by magnetic separation after the reaction was completed, and the product was washed, dried, and calcined, the calcination process was as follows: the temperature was raised to 480℃ at a temperature raising rate of 2.5℃ / min under a nitrogen atmosphere, and the temperature was kept for 2.5 h, to obtain the immobilized carrier;

[0101] S2, 95 g of the immobilized carrier in step S1 was added into 1.5 L of an ethanol aqueous solution (the mass fraction of ethanol was 75%), then 11 g of γ-glycidoxypropyltrimethoxysilane was added, and the mixture was reacted at 60℃ for 2.5 h, then the product was filtered, washed, and dried to obtain a pretreated immobilized carrier;

[0102] S3, modification of the immobilized carrier: 95 g of the pretreated immobilized carrier in step S2 was added into 1.5 L of a borate buffer with a pH of 9.5, then 7 g of L-lysine was added, and the mixture was reacted at 50℃ for 10 h, then the product was filtered, washed, and dried to obtain a modified immobilized carrier;

[0103] S4, 100 g of the modified immobilized carrier in step S3 was added into 1.5 L of a MES buffer with a pH of 5.5, then 25 g of EDC and 20 g of NHS were added, the mixture was stirred at 4℃ for 1.5 h, then the product was separated by a magnetic field after the activation was completed, and the product was washed to obtain an activated immobilized carrier; 100 g of the activated immobilized carrier was added into 2 L of a PBS buffer containing D-psicose 3-epimerase (the PBS buffer had a pH of 7.5, and the mass concentration of D-psicose 3-epimerase was 4 g / L), and the mixture was immobilized at 5℃ for 13 h, then the product was separated by a magnetic field after the immobilization was completed, and the product was repeatedly washed with the buffer until no enzyme activity was detected in the washing solution, to obtain the immobilized D-psicose 3-epimerase.

[0104] The preparation method of the composite additive is as follows:

[0105] By weight parts, 40 parts of glycerol, 0.8 parts of manganese sulfate, 15 parts of L-arginine are added into 1000 parts of deionized water, and after stirring uniformly, it is obtained.

[0106] The decolorization process in step (2) is: the D-allulose crude solution is pumped into a decolorization column filled with granular activated carbon, and is treated at a temperature of 50℃ and a flow rate of 3 BV / h; the filtration process is: the decolorized solution is sequentially passed through a precision filter and a 0.22 μm microporous filter membrane; the ion exchange process is: the clarified solution is sequentially passed through a strong acid cation exchange resin column (H + +) type and a weak base anion exchange resin column (OH - +) type in parallel, with temperature control at 25℃ and flow rate control at 4 BV / h; the nanofiltration process is: the solution after ion exchange is passed through a 150 Da nanofiltration membrane at 1 MPa to obtain a retentate; the chromatographic separation process is: with calcium ion (Ca 2+ +) type strong acid cation exchange resin as the stationary phase and deionized water as the mobile phase, the chromatographic separation temperature is controlled at 60℃.

[0107] Compared with Example 1, the composite additive in the present comparative example does not contain betaine.

[0108] Comparative Example 4

[0109] A preparation method of D-allulose, characterized in that it comprises the following steps:

[0110] (1) After D-fructose is prepared into a solution, it is sequentially subjected to activated carbon decolorization and ion exchange to obtain a refined D-fructose solution (mass concentration of 40%); the refined D-fructose solution is added into a batching tank, followed by addition of immobilized D-allulose 3-epimerase and a composite additive, the addition amount of the immobilized D-allulose 3-epimerase being 7% of the mass of the refined D-fructose solution, and the addition amount of the composite additive being 3% of the mass of the refined D-fructose solution, and isomerization reaction is carried out at 55-65℃ for 20 h, after which the immobilized enzyme is recovered from the reaction solution by magnetic separation to obtain a D-allulose crude solution;

[0111] (2) The D-allulose crude solution obtained in step (1) is subjected to decolorization, filtration, ion exchange, nanofiltration and chromatographic separation, the separated D-fructose dilute solution is concentrated and then used in step (1), and the D-allulose dilute solution is concentrated and then crystallized or dried to prepare D-allulose.

[0112] In step (1), the preparation method of the immobilized D-allulose 3-epimerase comprises the following steps:

[0113] S1, iron chloride and ferrous chloride were added into deionized water to obtain a mixed solution (the concentration of iron chloride was 0.5 mol / L, and the concentration of ferrous chloride was 0.3 mol / L), sodium citrate was added into the mixed solution, the molar ratio of sodium citrate to iron chloride was 0.35:1, and then the mixture was stirred uniformly, 25% ammonia water was added under a nitrogen atmosphere, the pH was adjusted to 11, and the mixture was aged at 75℃ for 1.5 h, then the magnetic Fe3O4 particles were separated by a magnetic field, and then the particles were washed and dried to obtain Fe3O4 particles; 95 g of the Fe3O4 particles were added into an ethanol aqueous solution (the mass ratio of ethanol to water was 8.5:1.5), then 80 g of hexadecyl trimethyl ammonium bromide, 350 g of 20% ammonia water, and 40 g of triisopropylbenzene were added, the mixture was stirred uniformly, then 180 g of tetraethyl orthosilicate was added, the mixture was stirred at 50℃ for 7 h, the product was collected by magnetic separation after the reaction was completed, and the product was washed, dried, and calcined, the calcination process was as follows: the temperature was raised to 480℃ at a temperature raising rate of 2.5℃ / min under a nitrogen atmosphere, and the temperature was kept for 2.5 h, to obtain the immobilized carrier;

[0114] S2, 95 g of the immobilized carrier in step S1 was added into 1.5 L of an ethanol aqueous solution (the mass fraction of ethanol was 75%), then 11 g of γ-glycidoxypropyltrimethoxysilane was added, and the mixture was reacted at 60℃ for 2.5 h, then the mixture was filtered, washed, and dried to obtain a pretreated immobilized carrier;

[0115] S3, modification of the immobilized carrier: 95 g of the pretreated immobilized carrier in step S2 was added into 1.5 L of a borate buffer with a pH of 9.5, then 7 g of L-lysine was added, and the mixture was reacted at 50℃ for 10 h, then the mixture was filtered, washed, and dried to obtain a modified immobilized carrier;

[0116] S4, 100 g of the modified immobilized carrier in step S3 was added into 1.5 L of a MES buffer with a pH of 5.5, then 25 g of EDC and 20 g of NHS were added, the mixture was stirred at 4℃ for 1.5 h, then the mixture was separated by a magnetic field after the activation was completed, and the activated immobilized carrier was obtained after being washed, 100 g of the activated immobilized carrier was added into 2 L of a PBS buffer containing D-psicose 3-epimerase (the PBS buffer had a pH of 7.5, and the mass concentration of D-psicose 3-epimerase was 4 g / L), and the mixture was immobilized at 5℃ for 13 h, then the immobilized D-psicose 3-epimerase was separated by a magnetic field after the immobilization was completed, and the immobilized D-psicose 3-epimerase was obtained by repeatedly washing with the buffer until no enzyme activity was detected in the washing solution.

[0117] The preparation method of the composite additive is as follows:

[0118] Add 40 parts of glycerol, 11 parts of betaine, and 0.8 parts of manganese sulfate into 1000 parts of deionized water, and stir until uniform, to obtain the product.

[0119] The decolorization process in step (2) is as follows: the D-allulose crude solution is pumped into a decolorization column containing granular activated carbon, and is treated at a temperature of 50℃ and a flow rate of 3 BV / h; the filtration process is as follows: the decolorized solution is sequentially passed through a precision filter and a 0.22 μm microporous filter membrane; the ion exchange process is as follows: the clarified solution is sequentially passed through a strong acid cation exchange resin column (H + +) type and a weak base anion exchange resin column (OH - +) type in parallel, with temperature control at 25℃ and flow rate control at 4 BV / h; the nanofiltration process is as follows: the solution after ion exchange is passed through a 150 Da nanofiltration membrane at 1 MPa to obtain a retentate; the chromatographic separation process is as follows: a strong acid cation exchange resin of calcium ion (Ca 2+ +) type is used as the stationary phase, and deionized water is used as the mobile phase, with chromatographic separation temperature control at 60℃.

[0120] In comparison with Example 1, the composite additive in the present comparative example is not added with L-arginine.

[0121] Comparative Example 5

[0122] A preparation method of D-allulose, characterized in that it comprises the following steps:

[0123] (1) D-fructose is prepared into a solution, and then sequentially subjected to activated carbon decolorization and ion exchange to obtain a refined D-fructose solution (mass concentration of 40%); the refined D-fructose solution is added into a batching tank, followed by addition of immobilized D-allulose 3-epimerase and a composite additive, the addition amount of the immobilized D-allulose 3-epimerase being 7% of the mass of the refined D-fructose solution, and the addition amount of the composite additive being 3% of the mass of the refined D-fructose solution, and isomerization reaction is carried out at 55-65℃ for 20 h, after which the immobilized enzyme is recovered from the reaction solution by magnetic separation to obtain a D-allulose crude solution;

[0124] (2) The D-allulose crude solution obtained in step (1) is subjected to decolorization, filtration, ion exchange, nanofiltration, and chromatographic separation, the separated D-fructose dilute solution is concentrated and then used in step (1), and the D-allulose dilute solution is concentrated and then subjected to crystallization or drying to obtain D-allulose.

[0125] In step (1), the preparation method of the immobilized D-allulose 3-epimerase comprises the following steps:

[0126] S1, iron chloride and ferrous chloride were added into deionized water to obtain a mixed solution (the concentration of iron chloride was 0.5 mol / L, and the concentration of ferrous chloride was 0.3 mol / L), sodium citrate was added into the mixed solution, the molar ratio of sodium citrate to iron chloride was 0.35:1, and then the mixture was stirred uniformly, 25% ammonia water was added under a nitrogen atmosphere, the pH was adjusted to 11, and the mixture was aged at 75℃ for 1.5 h, then the magnetic Fe3O4 particles were separated by a magnetic field, and then the particles were washed and dried to obtain Fe3O4 particles; 95 g of the Fe3O4 particles were added into an ethanol aqueous solution (the mass ratio of ethanol to water was 8.5:1.5), then 80 g of hexadecyl trimethyl ammonium bromide, 350 g of 20% ammonia water, and 40 g of triisopropylbenzene were added, the mixture was stirred uniformly, then 180 g of tetraethyl orthosilicate was added, the mixture was stirred at 50℃ for 7 h, the product was collected by magnetic separation after the reaction was completed, and the product was washed, dried, and calcined, the calcination process was as follows: the temperature was raised to 480℃ at a temperature raising rate of 2.5℃ / min under a nitrogen atmosphere, and the temperature was kept for 2.5 h, to obtain the immobilized carrier;

[0127] S2, 95 g of the immobilized carrier in step S1 was added into 1.5 L of an ethanol aqueous solution (the mass fraction of ethanol was 75%), then 11 g of γ-glycidoxypropyltrimethoxysilane was added, and the mixture was reacted at 60℃ for 2.5 h, then the product was filtered, washed, and dried to obtain a pretreated immobilized carrier;

[0128] S3, modification of the immobilized carrier: 95 g of the pretreated immobilized carrier in step S2 was added into 1.5 L of a borate buffer with a pH of 9.5, then 7 g of L-lysine was added, and the mixture was reacted at 50℃ for 10 h, then the product was filtered, washed, and dried to obtain a modified immobilized carrier;

[0129] S4, 100 g of the modified immobilized carrier in step S3 was added into 1.5 L of a MES buffer with a pH of 5.5, then 25 g of EDC and 20 g of NHS were added, the mixture was stirred at 4℃ for 1.5 h, then the product was separated by a magnetic field after the activation was completed, and the product was washed to obtain an activated immobilized carrier; 100 g of the activated immobilized carrier was added into 2 L of a PBS buffer containing D-psicose 3-epimerase (the PBS buffer had a pH of 7.5, and the mass concentration of D-psicose 3-epimerase was 4 g / L), and the mixture was immobilized at 5℃ for 13 h, then the product was separated by a magnetic field after the immobilization was completed, and the product was repeatedly washed with the buffer until no enzyme activity was detected in the washing solution, to obtain the immobilized D-psicose 3-epimerase.

[0130] The preparation method of the composite additive is as follows:

[0131] By weight parts, 11 parts of betaine, 0.8 parts of manganese sulfate, 15 parts of L-arginine are added into 1000 parts of deionized water, and after stirring uniformly, it is obtained.

[0132] The decoloring process in step (2) is that the D-allulose crude liquid is pumped into a decoloring column filled with granular activated carbon, and is treated at a temperature of 50°C and a flow rate of 3 BV / h; the filtering process is that the decolored liquid is sequentially passed through a precision filter and a 0.22 μm microporous filter membrane; the ion exchange process is that the clarified liquid is sequentially passed through a strong acid cation exchange resin column (H + type) and a weak base anion exchange resin column (OH - type) in parallel, with temperature control at 25°C and flow rate control at 4 BV / h; the nanofiltration process is that the ion-exchanged liquid is passed through a 150 Da nanofiltration membrane at 1 MPa to obtain a retentate; the chromatographic separation process is that a strong acid cation exchange resin of calcium ion (Ca 2+ ) type is used as the stationary phase, and deionized water is used as the mobile phase, with chromatographic separation temperature control at 60°C.

[0133] Compared with Example 1, the composite additive in the present comparative example does not contain glycerol.

[0134] The initial enzyme activity and the enzyme activity after 20 times of reuse of the immobilized D-allulose 3-epimerase prepared in Examples 1-3 and Comparative Examples 1-5 are tested respectively, and the conversion rate of allulose in Examples 1-3 and Comparative Examples 1-5 is calculated, and the results are shown in Table 1 below.

[0135] Enzyme activity determination of the immobilized D-allulose 3-epimerase: 0.1 g of the immobilized D-allulose 3-epimerase is taken, 0.1 mL of 50 mmol / L, pH 7.5 Tris-HCl buffer and 0.9 mL of 80 g / L fructose solution are added, and after incubation at 55°C water bath for 10 min, it is immediately transferred to boiling hot water to terminate the reaction for 5 min.

[0136] Enzyme activity definition: the amount of enzyme required to generate 1 μmol of D-allulose in 1 min under the above conditions is one enzyme activity unit.

[0137] Table 1

[0138]

[0139] As can be seen from Table 1 above, the immobilized D-allulose 3-epimerase prepared in the present application has good enzyme activity, high stability and excellent reuse, and the D-allulose preparation method provided in the present application has a high conversion rate, the purity of the product D-allulose is high, and has a good application prospect.

[0140] The above is further detailed description of the present application in combination with specific embodiments, and cannot be deemed as limitation of the specific embodiments of the present application. For those skilled in the art of the present application, some simple deductions or replacements can be made without departing from the concept of the present application, and all of them shall be deemed as falling within the protection scope of the present application.

[0141] Those skilled in the art can easily understand that the above description is only the preferred embodiments of the present application, and is not used to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for producing D-allulose, characterized by, The method comprises the following steps: (1) After D-fructose is prepared into a solution, active carbon decolorization and ion exchange are sequentially performed to obtain a refined D-fructose solution; the refined D-fructose solution is added into a batching tank, then immobilized D-psicose 3-epimerase and a composite additive are added, isomerization reaction is performed, after the reaction is completed, the immobilized enzyme is recovered from the reaction solution by magnetic separation to obtain a D-psicose crude solution; (2) The D-psicose crude solution obtained in step (1) is subjected to decolorization, filtration, ion exchange, nanofiltration and chromatographic separation, the separated D-fructose dilute solution is concentrated and then used in step (1), and the D-psicose dilute solution is concentrated and then subjected to crystallization or drying to prepare D-psicose. The preparation method of the immobilized D-psicose 3-epimerase in step (1) comprises the following steps: S1, iron chloride and ferrous chloride are added into deionized water to obtain a mixed solution, sodium citrate is added into the mixed solution, then ammonia water is added under a nitrogen atmosphere, the pH is adjusted, aging is performed to obtain Fe3O4 particles; the Fe3O4 particles are added into an ethanol aqueous solution, then cetyltrimethylammonium bromide, ammonia water and triisopropylbenzene are added, the mixture is uniformly stirred, then tetraethyl orthosilicate is added, stirring reaction is performed, the product is collected by magnetic separation, and the product is washed, dried and calcined to obtain an immobilized carrier; S2, the immobilized carrier is added into an ethanol aqueous solution, then γ-glycidoxypropyltrimethoxysilane is added, and heating reaction is performed to obtain a pretreated immobilized carrier; S3, the pretreated immobilized carrier is added into a borate buffer solution, then L-lysine is added, and constant-temperature reaction is performed to obtain a modified immobilized carrier; S4, the modified immobilized carrier is added into a MES buffer solution, then EDC and NHS are added, the mixture is stirred and activated, the mixture is washed and dried after magnetic separation to obtain an activated immobilized carrier; the activated immobilized carrier is added into a PBS buffer solution containing D-psicose 3-epimerase, and immobilization is performed, and the immobilized carrier is washed and dried after magnetic separation to obtain the immobilized D-psicose 3-epimerase; The preparation method of the composite additive is as follows: According to weight parts, 30-50 parts of glycerol, 8-13 parts of betaine, 0.5-1 part of manganese sulfate and 12-17 parts of L-arginine are added into 1000 parts of deionized water, and the mixture is uniformly stirred to obtain the composite additive.

2. The production method according to claim 1, characterized by, The mass concentration of the refined D-fructose solution in step (1) is 30-50%, the addition amount of the immobilized D-psicose 3-epimerase is 5-8% of the mass of the refined D-fructose solution, and the addition amount of the composite additive is 2-5% of the mass of the refined D-fructose solution.

3. The preparation method according to claim 1, characterized in that, In step S1, the concentration of iron chloride in the mixed solution is 0.3-0.8 mol / L, the concentration of ferrous chloride is 0.2-0.4 mol / L, the mass concentration of the ammonia water is 25-28%, the molar ratio of sodium citrate to iron chloride is 0.3-0.4:1, the pH is 10-11, the temperature of the aging is 70-80℃, and the time is 1-2 h.

4. The method of claim 1, wherein, The mass ratio of ethanol to water in the ethanol aqueous solution in step S1 is 8-9:1-2, the mass ratio of the Fe3O4 particles, cetyltrimethylammonium bromide, ammonia, triisopropylbenzene, and tetraethyl orthosilicate is 90-100:60-90:300-400:30-50:150-200, the temperature of the stirring reaction is 40-60℃, and the time is 5-8h, and the calcination process is as follows: heating to 450-500℃ at a heating rate of 2-3℃ / min under a nitrogen atmosphere, and keeping the temperature for 2-3h.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the immobilized carrier to γ-glycidoxypropyltrimethoxysilane in step S2 is 90-100:8-13, the temperature of the heating reaction is 55-65℃, and the time is 2-3h.

6. The method of claim 1, wherein, The pH of the borate buffer in step S3 is 9-10, the mass ratio of the pretreated immobilized carrier to L-lysine is 100:5-9, the temperature of the constant temperature reaction is 40-60℃, and the time is 8-12h.

7. The preparation method according to claim 1, characterized in that, The pH of the MES buffer in step S4 is 5-6, the mass ratio of the modified immobilized carrier to EDC to NHS is 100:15-30:10-25, the temperature of the stirring activation is 2-6℃, and the time is 1-2h; the pH of the PBS buffer is 7-8, the mass concentration of D-allulose 3-epimerase is 3-5g / L, the immobilization temperature is 4-6℃, and the time is 10-15h.

8. The method of claim 1, wherein, The temperature of the isomerization reaction in step (1) is 55-65℃, and the time is 15-20h.

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